| Part?Activation of iNOS/NO pathway and the over-expression of RhoABackground and objective:Rho GTPase, which is also called small GTP-binding protein, is a member of the large Rho family. Besides regulating actin, Rho GTPase is involved in many biological processes such as cell contraction, mobility, proliferation, apoptosis, and so on. Studies in recent years have shown that the RhoA/ROCK pathway is playing an important role in the initiation and development of many cardiovascular diseases. As an inflammatory factor that can initiate strong stimulatory effect, lipopolysaccharide (LPS) is involved in the pathological process ranging from atherosclerosis to intima thickening. The inducible nitric oxide synthase (iNOS) in vascular smooth cells (VSMCs) can be induced by lipopolysaccharide leading to the activation of iNOS/NO pathway. Part I of this study meant to identify the effect of LPS stimulation on the viability, proliferation of and RhoA expression level in VSMCs, meanwhile to clarify the role of iNOS/NO pathway in the regulation of RhoA by LPS.Methods:VSMCs were prepared from the thoracic aorta of Sprague-Dawley rats with explant culture, and the purity of cultured cells was confirmed by optical observation and immunohistochemistry.3-(4,5-dimethyl-2-thizolyl)-2,5-diphenyl-2H- tetrazolium bromide (MTT) assays were used to measure the viability of VSMCs under different LPS concentration. Immunohistochemistry counting was employed to evaluate the proliferation of LPS-treated cells. Activity of iNOS and NO production level were measured at different time-points after LPS stimulation. iNOS mRNA and protein expressions were analyzed by RT-PCR and immunoblot respectively. Analysis of RhoA protein expression at different time-points after LPS stimulation was performed. iNOS activity, NO production level, expression of iNOS protein and RhoA protein were respectively detected upon inhibition of iNOS/NO by iNOS inhibitor L-NIL or upon activation of iNOS/NO pathway by NO donor SNP.Results and conclusions:Morphological and immunohistochemistry evaluation confirmed the purity and eligibility of cultured VSMCs. LPS of 50?g/ml or lower dose didn't affect the viability of VSMCs. LPS of 25?g/ml and 50?g/ml exerted obvious promotive effect on the proliferation of VSMCs, suggesting a correlation of LPS with VSMCs-related proliferous diseases. LPS of 10?g/ml showed significant stimulatory effect on iNOS activity, NO production and iNOS expression which were increased in a time-dependent manner. The RhoA expression was also promoted by 10?g/ml LPS in a time-dependent fashion. L-NIL inhibited iNOS activity, decreased NO production and significantly suppressed the over-expression of RhoA. The over-expression of RhoA could also be induced by NO donor SNP, an effect similar to that exerted by LPS. These results implicated the iNOS/NO pathways in the induction of RhoA over-expression by LPS. Furthermore, increasing the dose of L-NIL achieved no stronger effect on down-regulating RhoA, implying that other pathways were also involved in the induction of RhoA by LPS.Part?Regulatory effects of acetylsalicylic acid on RhoABackground and objective:Acetylsalicylic acid (aspirin) is widely used in the treatment of vascular diseases. Studies in recent years have established a role of aspirin in suppressing smooth muscle cell proliferation, reducing inflammatory factors, protecting endothelial cells from the deleterious effects of oxidative stress. These multiple protective effects are not attributable to the platelet inhibitory action of aspirin and are cyclooxygenase (COX) independent. RhoA has been proved to be firmly correlated with the normal function and abnormal processes of cardiovascular system. This part of the study aimed to investigate the regulatory effects of aspirin on the aberrant expression of RhoA, also the protein activity, translocation and phosphorylation level of RhoA.Methods:MTT assay was employed to measure the viability of vascular smooth muscle cells and to determine the concentratin of aspirin used in our experiments. Real-time PCR and immunoblot analyses were performed to evaluate the regulatory effect of low-dose and high-dose aspirin on RhoA expression after LPS stimulation. The effect of low-dose and high-dose aspirin on regulating VSMCs RhoA activity was detected with pull-down assay. The translocation and phosphorylation level of RhoA proteins in non-stimulated or LPS-stimulated cells were analyzed respectively under treatment of low-dose or high-dose aspirin. The effect of low-dose and high-dose aspirin on LPS-induced VSMCs proliferation was investigated via immunohistochemistry counting. Results and conclusions:Results of the MTT experiment showed that aspirin of 300?mol/L and lower dose exerted no effects on the viability of VSMCs. Both low-dose (30?mol/L) and high-dose (300?mol/L) aspirin remarkably attenuated the LPS-induced elevation of RhoA mRNA and there was no difference between two aspirin groups. According to the analysis of protein expression, aspirin at a dose of 300?mol/L showed stronger suppressing effect versus 30?mol/L on the over-expressed RhoA protein. These results suggested that the COX-independent effect of aspirin originated, at least in part, from supressing RhoA over-expression and RhoA/ROCK pathway. Active RhoA (GTP-RhoA) increased by 80% after LPS-stimulation. The LPS-induced elevation of RhoA activity was not affected by low-dose aspirin but could be attenuated by high-dose aspirin. Translocation of RhoA protein was enhanced by LPS-stimulation, and neither low-dose nor high-dose aspirin caused significant change of translocation, implying that these proteins that involved in regulating RhoA translocation were not affected by aspirin. RhoA phosphorylation in LPS-treated VSMCs increased by 60%. Both low-dose and high-dose aspirin significantly suppressed the elevation of RhoA phosphorylation and the stronger suppressing effect was seen in high-dose aspirin group. Detection of proliferation manifested the inhibitory effect of high-dose aspirin on the LPS-enhanced proliferation, and no similar effect was found in group treated with low-dose aspirin.Part?Study on the mechanisms via which acetylsalicylic acid regulates RhoABackground and objective:As a member of the non-steroid anti-inflammatory drugs (NSAIDs), aspirin may inhibit cyclooxygenase and further inhibit the action of platelets. Many studies have proven that aspirin may exert multiple protective effects beside its anti-platelet function, but the underlying mechanisms of these multiple protective effects are largely unknown. Part II of this study showed that both low-dose and high-dose aspirin significantly down-regulated the over-expressed RhoA, so in Part III we aimed to investigate the underlying mechanisms through which aspirin exerted its down-regulating effect.Methods:iNOS activity, NO production and iNOS protein expression were detected to clarify the role of iNOS/NO pathway in conducting aspirin's effect. sGC/cGMP/PKG pathway was inhibited and activated with the presence of aspirin treatment to analyze the involvement of this pathway in LPS-induced RhoA over-expression, and to analyze the correlation of this pathway with the down-regulating effect of aspirin. Comparison among aspirin, salicylate and indomethacin was conducted to make clear whether the down-regulation of RhoA was associated with acetylation and cyclooxygenase. Effects of aspirin on the stability of RhoA were studies via evaluating the half-life of RhoA mRNA and RhoA protein. The ratio of GTP-RhoA during degradation was measured and the influence of translocation and phosphorylation on RhoA degradation were analyzed.Results and conclusions:The LPS-induced elevation of iNOS activity, NO production and iNOS protein expression was not affected by low-dose aspirin; High-dose aspirin was able to decrease iNOS activity and NO production, but not the expression of iNOS protein, suggesting that the inhibition (by high-dose aspirin) on iNOS activity is one of the mechanisms of aspirin's multiple protective effects, but not the major mechanism through which aspirin suppressed the over-expression of RhoA. The inhibition of sGC/cGMP/PKG pathway by ODQ (a specific inhibitor of soluble guanylyl cyclases) led to the down-regulation of LPS-induced RhoA, while this inhibitory effect of ODQ was reversed by 8-Br-PET-cGMP, a cGMP analog. Treatment with 8-Br-PET-cGMP elicited an up-regulation of RhoA mRNA and protein which was similar to that induced by LPS. The up-regulation of RhoA elicited by 8-Br-PET-cGMP was abrogated by the addition of aspirin (30?mol/L), implying that sGC/cGMP/PKG and its downstream effectors are the targets of aspirin. Comparative analyses showed that aspirin was the only one being able to suppress the over-expressed RhoA, suggesting that the regulatory effect of aspirin is acetylation-dependent. Aspirin exerted no effect on the half-life of RhoA mRNA but significantly shortened the half-life of RhoA protein, demonstrating that the attenuation of RhoA over-expression was conducted both through down-regulating RhoA mRNA and through enhancing RhoA protein degradation. The ratio of GTP-RhoA was relatively increased during RhoA protein degradation. Phosphorylation and translocation analyses proved that these two degradation-preventive factors were not responsible for the relative elevation of GTP-RhoA, suggesting a susceptibility of GDP-RhoA to the aspirin-enhanced degradation. |
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