Rho激酶在心血管疾病中新进展及Rho激酶抑制剂的应用

Rho激酶(ROCK)被认为是治疗心血管疾病的新靶点。本文主要简述RhoA/ROCK通路在调节脉管系统的功能,并探讨其在心血管疾病发病中的作用机制。     

Rho/Rho kinase is a key enzyme system involved in the angiotensin II signaling pathway of liver fibrosis and steatosis

BACKGROUND AND AIM: The molecular mechanisms underlying the involvement of the renin-angiotensin system in hepatic fibrosis are unclear. Recently, it was reported that a Rho kinase inhibitor prevented fibrosis of various tissues and that the Rho/Rho kinase pathway was involved in the renin-angiotensin system of vascular smooth muscle cells. In this study, the involvement of the Rho/Rho kinase pathway on angiotensin II signaling in liver fibrogenesis and generation of steatosis was investigated. METHODS: Rats were fed a choline-deficient/L-amino acid-defined (CDAA) diet continuously and treated with a Rho kinase inhibitor, Y-27632, and an angiotensin II receptor blocker, TCV-116. Liver histology and hepatic stellate cell activation were analyzed. Free radical production was detected by 4-hydroxynonenal and 8-hydroxy-2'-deoxyguanosine immunostaining and the expression of tumor necrosis factor-alpha was examined. Isolated hepatic stellate cells were pretreated with a Rho kinase inhibitor, Y-27632, or an angiotensin II receptor blocker, CV-11974, and stimulated with angiotensin II, and mRNA expression of transforming growth factor-beta and alpha-smooth muscle actin was analyzed. RESULTS: Both the angiotensin II receptor blocker and the Rho kinase inhibitor improved fibrosis and steatosis of the liver in CDAA-fed rats. The increase in the number of hepatocytes positive for 4-hydroxynonenal and 8-hydroxy-2'-deoxyguanosine in CDAA-fed rats was significantly prevented by the angiotensin II receptor blocker and the Rho kinase inhibitor. The levels of tumor necrosis factor-alpha mRNA in the liver of CDAA-fed rats were significantly increased and this increase was significantly inhibited by treatment with the angiotensin II receptor blocker and the Rho kinase inhibitor. mRNA expression of transforming growth factor-beta and alpha-smooth muscle actin stimulated by angiotensin II was also significantly suppressed by these two drugs. CONCLUSION: These results suggest that the Rho/Rho kinase pathway is at least partly involved in the renin-angiotensin system and plays an important role in hepatic fibrosis and steatosis.     

Role of endothelin in diabetic vascular complications

Endothelin-1 (ET-1), a 21 amino acid peptide originally purified from conditioned medium of cultures of porcine aortic endothelial cells, is recognized as a product of many other cells as well. It is now known that there are three endothelin genes in the human genome (ET-1, ET-2, and ET-3). ET-1 and ET-2 are both strong vasoconstrictors, whereas ET-3 is a potentially weaker vasoconstrictor than the other two isoforms. Besides being the most potent vasoconstrictor yet known, ET-1 also acts as a mitogen on the vascular smooth muscle, and, thus, it may play a role in the development of vascular diseases. It is well known that accelerated angiopathy is a major complication in diabetes mellitus. As generalized endothelial cell damage is thought to occur in diabetic patients, ET-1, being released from the damaged endothelial cells, is able to make contact with the underlying vascular smooth muscle cells and thus could be one important cause of diabetic angiopathy. This article summarizes the reported literature of the role of ET-1 in the development of diabetic complications, with particular focus on the possible role of Et-1 in mediating the effects of angiotensin-converting enzyme inhibitors.     

Rho激酶抑制剂法舒地尔在冠心病治疗中的应用进展

Rho激酶参与了冠心病发病的病理生理机制,Rho激酶抑制剂具有抗血管痉挛和抗动脉硬化等作用.因此,Rho激酶抑制剂有望成为治疗冠心病的新药物.本文就Rho激酶抑制剂法舒地尔在冠心病治疗中的应用进展作一综述.     

支气管哮喘患者Rho激酶信号通道与T调节细胞的相关性

支气管哮喘（简称哮喘）被认为是一种气道慢性非特异性炎症性疾病,许多细胞因子和炎症介质参与了这个病理生理过程。目前研究显示,Rho激酶信号通道及T调节细胞在哮喘的发病机制中起重要作用,而他汀类药物也具有抑制促炎性细胞因子和细胞因子的分泌,增加调节性T细胞数量和功能等作用。本文就哮喘中Rho激酶信号转导通路与调节T细胞的相关性进行简要综述。     

系统性红斑狼疮患者外周血T细胞Rho激酶活性检测及其意义

目的 研究系统性红斑狼疮(SEE)患者外周血T细胞中Rho激酶(ROK)活化情况,并进一步探讨其临床意义.方法 收集SLE患者30例,健康人12名和类风湿关节炎(RA)患者8例作为对照组.外周血T细胞分离采用Rosettsep T细胞提取试剂盒提取,ROK活性用磷酸化MYPT1蛋白表达来表示,磷酸化MYPT1蛋白检测采用免疫印迹法,T细胞增殖采用四甲基偶氮唑蓝比色(MTT)法,细胞因子水平采用酶联免疫吸附试验(ELISA)检测.结果 新鲜分离的SLE患者外周血T细胞ROK活性均显著高于健康对照组和RA组;SLE患者T淋巴细胞在体外培养24 h后ROK活性与健康对照组差异无统计学意义;ROK特异性抑制剂Y27632显著抑制SLE患者T淋巴细胞增殖以及分泌自细胞介素(IL)-6,但对IL-10分泌无显著影响.中、重度病情活动的SLE患者外周血T淋巴细胞ROK活性显著高于轻度活动者(P均＜0.05).SLE患者关节炎、神经精神症状和蛋白尿表现阳性组T淋巴细胞ROK活性显著高于上述临床表现阴性组.结论 SLE患者T淋巴细胞存在ROK信号活化异常,并且可能与T淋巴细胞活化、狼疮? 疾病活动性和某些脏器损害有关.     

Protein kinase C and the development of diabetic vascular complications.

Hyperglycemic control in diabetes is key to preventing the development and progression of vascular complications such as retinopathy, nephropathy and neuropathy. Increased activation of the diacylglycerol (DAG)-protein kinase C (PKC) signal transduction pathway has been identified in vascular tissues from diabetic animals, and in vascular cells exposed to elevated glucose. Vascular abnormalities associated with glucose-induced PKC activation leading to increased synthesis of DAG include altered vascular blood flow, extracellular matrix deposition, basement membrane thickening, increased permeability and neovascularization. Preferential activation of the PKCbeta isoform by elevated glucose is reported to occur in a variety of vascular tissues. This has lead to the development of LY333531, a PKCbeta isoform specific inhibitor, which has shown potential in animal models to be an orally effective and nontoxic therapy able to produce significant improvements in diabetic retinopathy, nephropathy, neuropathy and cardiac dysfunction. Additionally, the antioxidant vitamin E has been identified as an inhibitor of the DAG-PKC pathway, and shows promise in reducing vascular complications in animal models of diabetes. Given the overwhelming evidence indicating a role for PKC activation in contributing to the development of diabetic vascular complications, pharmacological therapies that can modulate this pathway, particularly with PKC isoform selectivity, show great promise for treatment of vascular complications, even in the presence of hyperglycemia.     

Limited joint mobility in type 1 diabetic patients: correlation to other diabetic complications

Abstract. Objectives. To examine the association between limited joint mobility (LJM) and complications of diabetes in adult patients with type 1 diabetes. Design. Cross-sectional study in diabetic patients and healthy controls. Setting. The study was performed at the department of medicine in Turku University Hospital (n = 103), a private diabetes outpatient clinic (n = 153) and the municipal health centre of Turku (n = 29), Finland. Subjects. We studied 285 diabetic patients [age (mean ± SD): 33.4 ± 10.0 years] and 288 healthy nondiabetic controls [age (mean ± SD): 32.3 ± 9.2]. Main outcome measures. The limitations of several joints were examined with a goniometer. The diabetic patients were assessed in terms of the following complications: background and proliferative retinopathy, peripheral symmetrical polyneuropathy, autonomic neuropathy, impotence as well as clinical and incipient nephropathy; serum lipid values were also measured. Results. The prevalences of LJM were 58% and 14% in diabetic patients and in healthy controls, respectively. The diabetic patients with LJM had a 2.8-fold risk of proliferative retinopathy [95% confidence interval (CI): 1.1–7.3] and a 3.6-fold risk of nephropathy (95% CI: 1.4–9.3) compared to patients without LJM, when the confounding effect of the duration of diabetes was excluded. LJM was not related to metabolic control of diabetes, microalbuminuria, autonomic neuropathy or impotence. The association between LJM and peripheral symmetrical polyneuropathy was exclusively explained by the duration of diabetes. The correlation between LJM and serum total and low-density lipoprotein cholesterol was dependent on the association between LJM and nephropathy. LJM did not relate to serum high-density lipoprotein cholesterol or triglyceride values. Conclusions. The diabetic patients with LJM had an increased risk of proliferative retinopathy and nephropathy compared to patients without LJM.     

Blockade of Rho/Rho-associated coiled coil-forming kinase signaling can prevent progression of hepatocellular carcinoma in matrix metalloproteinase-dependent manner

Aim:  There is growing evidence that the Rho/Rho-associated coiled coil-forming kinase (ROCK) signaling pathway is upregulated in tumors and plays a key role in cancer invasion and metastasis. Our aim was to test the anticancer effects of Rho/ROCK inhibitor, Y-27632, including possible mechanisms in a highly-metastasizing hepatocellular carcinoma (HCC) mouse model on its secretion of matrix metalloproteinase (MMP) and tumor progression.
Methods:  Following orthotopic implantation of CBO140C12 HCC tumor fragments into the liver of mice, the mice were randomly assigned to a Y-27632-treated group or control group. After treatment for 4 weeks, specimens were obtained to evaluate tumor size, metastases, and immunohistochemical findings. In vitro , we examined the effects of Y-27632 and RhoC siRNA on MMP-2 and -9 expressions, invasiveness, and apoptosis in cultured tumor cells.
Results:  Both RhoA and RhoC were upregulated in HCC-bearing livers, and Y-27632 significantly inhibited not only tumor growth and intrahepatic metastasis ( P  < 0.05), but also tumoral MMP-9 expression. Moreover, Y-27632 treatment resulted in large necrotic areas in tumors. In vitro , Y-27632 and RhoC siRNA reduced MMP-2 and -9 expressions, as well as the chemotactic migration of tumor cells dose-dependently, and increased apoptosis eight times.
Conclusion:  Y-27632 suppresses progression and limits the intrahepatic metastasis of established HCC. This could be linked to the decreased MMP expression and induction of apoptosis in tumor cells. Rho signaling may prove to be a productive target in anticancer therapy.     

Chronic inhibition of Rho kinase blunts the process of left ventricular hypertrophy leading to cardiac contractile dysfunction in hypertension-induced heart failure

The Gq-RhoA-Rho kinase pathway, activated by neurohormonal factors such as angiotensin II (Ang II), has been proposed to be one of the important signaling pathways involved in the progression of left ventricular (LV) hypertrophy to heart failure. We tested the hypothesis that chronic inhibition of Rho kinase prevents this process. Heart failure was induced in Dahl salt-sensitive (DS) rats fed an 8% NaCl diet from 8 until 17 weeks of age. Y-27632 (5 mg/kg per day), a selective Rho kinase inhibitor, was applied orally to DS rats starting at 10 weeks of age for 7 weeks (DS/Y+). DS rats without Y-27632 (DS/Y-) and Dahl salt-resistant (DR) rats fed the 8% NaCl diet were regarded as non-therapeutic and normotensive controls, respectively. At 17 weeks of age, there was no significant difference in the blood pressure of DS/Y- and DS/Y+ rats. DS/Y- rats exhibited: (1) increases in LV mass, cross-sectional area (CSA) of cardiomyocytes, and interstitial fibrosis; (2) contractile dysfunction, i.e. decreases in LV ejection fraction and % fractional shortening, and prolongation of time to peak tension as well as to 50% relaxation in the twitch contraction of isolated papillary muscle; and (3) increases in the protein expression of Galphaq and Rho kinase in the myocardial membrane fraction. In DS/Y+ rats, the degree of myocardial hypertrophy was significantly inhibited in association with improved contractile function, without a decrease in the degree of interstitial fibrosis. Our results suggest the possibility that the Gq-Rho kinase pathway plays an important role in the process of hypertension-induced LV hypertrophy leading to contractile dysfunction.     

Involvement of Rho kinase (ROCK) in sepsis-induced acute lung injury

Indirect acute lung injury is associated with high morbidity and mortality. We investigated the link between Rho kinase (ROCK) activation and apoptotic cell death in sepsis induced acute lung injury. This hypothesis was tested by administering a specific, selective inhibitor of ROCK (Y-27632) to rats subjected to cecal ligation and puncture (CLP). Rats were randomly divided into 4 groups as; sham-operated, sham + Y-27632, CLP and CLP + Y-27632. Twenty-four hours later, each experiment was terminated and lungs analyzed. Histopathology was assessed by hematoxylin-eosin staining and the presence of apoptosis was evaluated through the TUNEL assay. Pulmonary activity of caspase 3 and ROCK 1 & 2 were measured by western blot. Interstitial edema, severely damaged pulmonary architecture with massive infiltration of the inflammatory cells and an increase in lung tissue TBARS levels as well as 3-NT to total tyrosine ratios were observed in untreated CLP animals. Pretreatment of animals with Y-27632, reduced lung injury in the CLP induced septic rats in each of these parameters of lung injury (p<0.05). Western immunoblot revealed active caspase cleavage and increased expression of active fragment of ROCK 1 & 2 in the CLP group. TUNEL assay showed an increase in percentage of apoptotic cells when comparing the CLP group with the CLP + Y-27632 group. These results suggest an important role of Rho kinase in sepsis induced lung injury by a mechanism that might be related to oxidative and/or nitrosative stress mediated caspase cleavage leading to apoptosis.     

Association between diabetic eye disease and other complications of diabetes: Implications for care. A systematic review

The aim of this systematic review was to examine the associations between diabetic retinopathy (DR) and the common micro- and macrovascular complications of diabetes mellitus, and how these could potentially affect clinical practice. A structured search of the PubMed database identified studies of patients with diabetes that assessed the presence or development of DR in conjunction with other vascular complications of diabetes. From 70 included studies, we found that DR is consistently associated with other complications of diabetes, with the severity of DR linked to a higher risk of the presence of, or of developing, other micro- and macrovascular complications. In particular, DR increases the likelihood of having or developing nephropathy and is also a strong predictor of stroke and cardiovascular disease, and progression of DR significantly increases this risk. Proliferative DR is a strong risk factor for peripheral arterial disease, which carries a risk of lower extremity ulceration and amputation. Additionally, our findings suggest that a patient with DR has an overall worse prognosis than a patient without DR. In conclusion, this analysis highlights the need for a coordinated and collaborative approach to patient management. Given the widespread use of DR screening programmes that can be performed outside of an ophthalmology office, and the overall cost-effectiveness of DR screening, the presence and severity of DR can be a means of identifying patients at increased risk for micro- and macrovascular complications, enabling earlier detection, referral and intervention with the aim of reducing morbidity and mortality among patients with diabetes. Healthcare professionals involved in the management of diabetes should encourage regular DR screening.     

蛋白激酶C抑制剂改善帕金森病大鼠异动症的研究

目的 探讨蛋白激酶C(PKC)抑制剂他莫昔芬对左旋多巴诱发异动症的细胞学与行为学效应. 方法将6-羟多巴胺立体定向注射于大鼠前脑内侧束建立帕金森病(PD)动物模型.模型成功的PD大鼠接受每天2次左旋多巴(50 mg/kg左旋多巴+12.5 mg/kg苄丝肼)腹腔注射,持续22 d.在第23天左旋多巴注射前,2组PD大鼠给予他莫昔芬或溶剂处理.评估旋转反应时间及剂峰旋转次数,采用蛋白印迹法检测纹状体区N-甲基-D-天冬氨酸受体NR1亚单位(NR1)和890、896位丝氨酸位点磷酸化的NR1(NR1S890、NR1S896)表达情况. 结果 PKC抑制剂他莫昔芬减轻少了左旋多巴诱导的PD大鼠剂峰旋转次数[(121.0±7.6)次与(161.5±22.1)次,t=2.6,P<0.05];此外,他莫昔芬能调节与异动症密切相关的NR1的亚细胞分布,使纹状体部位NR1在膜蛋白中的表达量减少至(82.4±5.1)%,与左旋多巴+溶剂组[(103.0±3.5)%]比较差异有统计学意义(t=6.7,P<0.05),同时使NR1S890、NR1S896的磷酸化水平明显降低,其表达量分别为(77.2±4.2)%与(98.4±6.4)%(t=5.9、3.6,均P<0.05). 结论长期左旋多巴作用激活了纹状体棘状神经元内丝氨酸激酶PKC,并导致谷氨酸受体磷酸化,从而出现了异动症;抑制丝氨酸激酶(如PKC)活性的药物可能是治疗及预防帕金森病异动症的一种新的治疗方式.     

住院老年2型糖尿病患者微血管病变的相关因素分析

目的探讨老年2型糖尿病患者微血管病变的构成比及相关因素。方法用回顾性分析的方法研究2003年～2010年于卫生部北京医院住院治疗的年龄≥60岁的2型糖尿病患者876例,分为糖尿病肾病(DN)组和非糖尿病肾病(非DN)组,糖尿病视网膜病变(DR)组和非糖尿病视网膜病变(非DR)组,糖尿病周围神经病变(DPN)组和非糖尿病周围神经病变(非DPN)组,计算DN、DR、DPN构成比,比较患者的临床特点,并探寻老年2型糖尿病患者DR、DN、DPN的相关因素。结果 (1)DN构成比为34.5%,DR构成比为42.4%,DPN构成比为82.3%。(2)DN与非DN两组间体质量指数(BMI)、糖尿病病程、高血压病程、收缩压(SBP)、舒张压(DBP)、空腹血糖(FBS)、糖化血红蛋白(HbA1c)、空腹胰岛素(Fins)、高密度脂蛋白胆固醇(HDL)、甘油三酯(TG)、尿酸(UA)均有显著性差异(P<0.05或P<0.01);DR与非DR两组间仅糖尿病病程、SBP、空腹C肽(FCP)有显著性差异(均P<0.01);DPN与非DPN两组间年龄、糖尿病病程、HbA1c、TC、LDL有显著性差异(P<0.05或P<0.01)。(3)Logistic回归结果显示,DN与SBP、HbA1c、FBS、HDL、UA、糖尿病病程有关(OR值分别为1.022、1.098、1.075、0.501、1.004,1.048,P<0.05或P<0.01);DR与SBP、HbA1c、糖尿病病程有关(OR值分别为1.017、1.102、1.097,P<0.05或P<0.01);DPN与HbA1c、LDL、糖尿病病程、年龄有关(OR值分别为1.226、1.370、1.041、1.058,P<0.05或P<0.01)。结论对于老年2型糖尿病患者,DN、DR、DPN均与糖尿病病程和HbA1c有关,控制血糖对防治微血管病变意义重大,综合控制血糖、血压、血脂、尿酸可以更好的防治糖尿病微血管并发症。     

模拟失重大鼠胸主动脉收缩功能的变化可能与Rho激酶改变有关

目的:观察模拟失重大鼠胸主动脉收缩功能的变化及Rho相关的蛋白激酶（Rho-associated protein kinase,ROCK）表达的改变,并探讨二者之间的关系。方法: 以尾部悬吊4周建立模拟失重大鼠模型并观察模拟失重对胸主动脉的主要生理的影响。采用离体血管环功能实验检测大鼠胸主动脉的收缩反应性变化;通过蛋白印迹技术检测大鼠胸主动脉ROCK II蛋白的表达。结果: 与对照组相比,悬吊组氯化钾、苯肾上腺素诱导的大鼠胸主动脉收缩功能均明显增强（P<0.05）。用ROCK特异性抑制剂Y-27632孵育1 h后,两组胸主动脉的收缩反应均显著降低至同一水平,两组间无统计学差异。蛋白印迹结果显示,悬吊组ROCK II的表达增加。结论: ROCK表达的改变可能在模拟失重大鼠胸主动脉收缩功能增强中发挥重要作用,去除ROCK的作用可消除模拟失重大鼠与正常大鼠胸主动脉收缩功能的差异。     

Relation of diabetic control to development of microvascular complications

Summary This review seeks to supply the arguments which support or deny the relationship between the quality of control of blood glucose levels and the course of diabetic microangiopathy. The ideal study is impossible to do in man but most prospective studies suggest that the better the control the slower the rate of progression and severity of lesions. Scientific but indirect arguments from biochemical, enzymatic and functional studies have shown that insulin and/or blood glucose control reverse some early diabetic changes that are probably related to the late events. Recent studies suggest that observations on animals that have shown the beneficial effect of treatment are relevant to the problem of diabetic microvascular lesions in man. Heredity influences the development of diabetic microangiopathy in diabetics but retinal and/or glomerular lesions — which are definitely not pathognomonic for diabetes — do not appear in the absence of hyperglycaemia. Muscle capillary basement membrane thickening that seems not to be a specific abnormality was not observed by several investigators in recently diagnosed diabetics. Therefore most of the arguments that have been given to support the point of view that tight control is not justified may be rejected. The opinion of the author is that strict control of diabetes is worthwile in patients with long life expectancy and no psychological, social or cultural handicaps.     

Expert opinion on thyroid complications of new anti-cancer therapies: Tyrosine kinase inhibitors

Thyroid pathology is the most frequent form of endocrinopathy during tyrosine kinase inhibitor (TKI) treatment. Dysthyroidism occurs in 10% to 80% of cases, depending on diagnostic criteria. In patients with intact thyroid gland prior to TKI treatment, incidence of dysthyroidism is 30–40%, with subclinical presentation in half of cases. It mainly involves hypothyroidism, preceded in 20–40% of cases by transient thyrotoxicosis that may go overlooked. The pathophysiological mechanism is “vascular” thyroiditis induced by the anti-angiogenic action of TKIs. Between 20% and 60% of patients receiving levothyroxine ahead of TKI treatment show increased levothyroxine requirements. TKIs should not be discontinued because of onset of thyroid dysfunction. Treatment is symptomatic in case of thyrotoxicosis, and levothyroxine replacement therapy is initiated in case of symptomatic hypothyroidism or TSH > 10 mIU/L. During TKI treatment, TSH should be assayed monthly, or at end of off-period (i.e., day 1 of new cycle after interruption), for the first 6 months, then every 2–3 months or in case of clinical signs of dysthyroidism. In patients already treated for hypothyroidism, TSH should be assayed monthly for 3 months, then every 3 months throughout treatment. At TKI termination, remission of hypothyroidism is possible but unpredictable, and progressive discontinuation of levothyroxine may be considered under monitoring. Teamwork between oncologists and endocrinologists improves screening and treatment of thyroid dysfunction, enabling the patient to be better accompanied during treatment.