Living high training low induces physiological cardiac hypertrophy accompanied by down-regulation and redistribution of the renin-angiotensin system

Aim:

Living high training low” (LHTL) is an exercise-training protocol that refers living in hypoxia stress and training at normal level of O₂. In this study, we investigated whether LHTL caused physiological heart hypertrophy accompanied by changes of biomarkers in renin-angiotensin system in rats.

Methods:

Adult male SD rats were randomly assigned into 4 groups, and trained on living low-sedentary (LLS, control), living low-training low (LLTL), living high-sedentary (LHS) and living high-training low (LHTL) protocols, respectively, for 4 weeks. Hematological parameters, hemodynamic measurement, heart hypertrophy and plasma angiotensin II (Ang II) level of the rats were measured. The gene and protein expression of angiotensin-converting enzyme (ACE), angiotensinogen (AGT) and angiotensin II receptor I (AT₁) in heart tissue was assessed using RT-PCR and immunohistochemistry, respectively.

Results:

LLTL, LHS and LHTL significantly improved cardiac function, increased hemoglobin concentration and RBC. At the molecular level, LLTL, LHS and LHTL significantly decreased the expression of ACE, AGT and AT₁ genes, but increased the expression of ACE and AT₁ proteins in heart tissue. Moreover, ACE and AT₁ protein expression was significantly increased in the endocardium, but unchanged in the epicardium.

Conclusion:

LHTL training protocol suppresses ACE, AGT and AT1 gene expression in heart tissue, but increases ACE and AT₁ protein expression specifically in the endocardium, suggesting that the physiological heart hypertrophy induced by LHTL is regulated by region-specific expression of renin-angiotensin system components.     

Expression of the renin and angiotensinogen genes

Recent advances in molecular biological techniques provide us with genetic approaches for studying the renin-angiotensin system. Renin and angiotensinogen cDNAs have been cloned in several species, and the sequences are highly conserved between the species. The 5'-flanking region of the human renin gene indicated putative regulatory sequences of glucocorticoid, estrogen, progesterone, and cAMP. The 5'-flanking region of the human angiotensinogen gene also had putative regulatory sequences of glucocorticoid, estrogen, acute phase protein, and cAMP. These structures may be related to the tissue specific expression of the renin and angiotensinogen genes. In this review, expression of rat renin and angiotensinogen genes in various tissues in the following conditions are described: a) different sodium intake in the liver, kidney, and brain; b) angiotensin II and converting enzyme inhibition in the liver, kidney and brain; c) renovascular hypertension in the kidney and liver; d) aging in the liver and kidney; e) adrenal steroids in the liver, kidney and brain; f) gonadotropin and testosterone in the testes, liver and kidney; g) triiodothyronine in the liver, kidney and brain; h) nephrectomy in the liver and brain; i) high potassium, angiotensin II, sodium intake and nephrectomy in the adrenal gland; j) transgenic animal. Our results suggest that the expression of the renin and angiotensinogen genes are regulated in a tissue-specific manner.     

钾通道基因Kv 1.5在大鼠脑不同发育阶段的mRNA表达研究

目的　研究大鼠大脑不同发育阶段钾通道基因Ｋｖ１－５的ｍＲＮＡ表达。方法　①利用一步法提取脑组织中ＲＮＡ;②ＤＮＡ模板的制备;③体外转录合成ＲＮＡ探针;④ＲＮＡ：ＲＮＡ杂交。结果　Ｋｖ１－５在大鼠胎儿期大脑皮层未表达,出生２５ｄ后表达明显增强,成年大鼠与出生后２５ｄ相比表达未发生变化。结论　Ｋｖ１－５ｍＲＮＡ的表达与发育阶段有较大关系,出生前期Ｋｖ１－５ｍＲＮＡ表达快速增长可能与大鼠脑发育和功能分化有关。     

Cross talk of tumor necrosis factor-alpha and the renin-angiotensin system in tumor necrosis factor-alpha-induced plasminogen activator inhibitor-1 production from hepatocytes

Tumor necrosis factor (TNF)-alpha and local activation of the renin-angiotensin system may contribute to insulin resistance and atherosclerosis. In this study, we investigated the involvement of these mediators in the liver. We found that the gene expression of renin-angiotensin system components, together with that of plasminogen activator inhibitor (PAI)-1, is upregulated in the liver of patients with obesity and type 2 diabetes. We next examined the role of the renin-angiotensin system on TNF-alpha-induced PAI-1 production in the nonmalignant human hepatocyte cell line THLE-5b. THLE-5b cells expressed genes encoding renin-angiotensin system components including angiotensinogen, angiotensin-converting enzyme (ACE), and angiotensin type 1 (AT(1)) receptor. ACE, angiotensinogen, and angiotensin AT(1) receptor mRNA expression were upregulated time-dependently by TNF-alpha. Moreover, angiotensin AT(1) receptor antagonist dose-dependently inhibited TNF-alpha-induced PAI-1 production. Interestingly, high-dose olmesartan, but not candesartan, reduced the increased expression of the angiotensin AT(1) receptor. These results suggest that TNF-alpha and the local renin-angiotensin system coordinately stimulate PAI-1 production in hepatocytes. Selective angiotensin AT(1) receptor antagonists inhibit both TNF-alpha- and angiotensin II-induced PAI-1 production in hepatocytes, suggesting a cross talk between both systems.     

Quantitation of dihydropyrimidine dehydrogenase (DPD) mRNA expression levels in normal colon and colorectal cancer tumor paraffin-embedded tissue specimens

5-Fluorouracil (5-FU) has been used for more than 40 years in the treatment of neoplastic disease, and remains the standard first-line treatment for colorectal cancer in combination with irinotecan and leucovorin. Previous studies indicated that measurement of dihydropyrimidine dehydrogenase (DPD) gene expression before treatment was valuable in determining the potential benefit of and toxicity to 5-FU treatment. In this study, we investigated the association between intratumoral DPD gene expression and the adjacent normal tissue DPD gene expression and DPD mRNA expression level in non-paired colon tumor and normal colon tissue specimens. In addition, we have compared the difference of DPD gene expression at three different RNA concentrations from the same specimen (180, 100 and 5 ng/reaction, respectively). DPD expression was measured by quantitative RT-PCR using a LightCycler instrument in a total of 31 specimens. Gene expression values were expressed as a ratio of target gene (DPD) to the internal reference gene (G6PDH). Our study revealed no statistically significant difference (p=0.23) between tumor tissues and matched normal tissue in DPD expression. In contrast, the data on DPD mRNA expression in non-paired colon tumor and normal tissue specimens revealed a significant difference (p=0.0004) between the tumor group and the normal group. In the three RNA concentration groups, there was no significant difference (p=0.55) in gene expression at the different RNA concentrations from the same donor. These results demonstrate that intratumoral gene expression levels of DPD do not correlate with tumor cell percentage or with RNA concentration. Thus, DPD mRNA expression appears to be a valid sensitivity test for 5-FU in spite of a varying density of tumor cells and RNA yield in specimens submitted for analysis.     

Effect of berberine on PPARα-NO signalling pathway in vascular smooth muscle cell proliferation induced by angiotensin IV

Context: The available treatments for the abnormal proliferation of vascular smooth muscle cells (VSMCs) are still dismal. Berberine has been demonstrated to possess extensive medicine activity, yet relatively little is known about its effect on VSMCs proliferation. Many studies showed that PPARα and NO participated in the process of VSMCs proliferation.

Objective: To evaluate the effect of berberine and its possible influence on PPARα-NO pathway in angiotensin IV-stimulated VSMCs.

Materials and methods: The primary VSMCs were cultured with the tissue explants method, and the proliferation was characterized by MTT and protein content. Protein and mRNA expression were measured by Western blot and real-time RT-PCR, respectively. NO synthase (NOS) activity was measured using a spectrophotometric assay, and NO concentration was measured using the Griess assay.

Results: Angiotensin IV (0.1?nmol/L)-induced VSMCs proliferation was evidenced by increasing the optical density at A₄₉₀ and total protein content (p?p?p?p?p?p?Discussion and conclusion: The results support the therapeutic effects of berberine on angiotensin IV-induced proliferation in cultured VSMCs at least partially through targeting the PPARα-NO signalling pathway.     

Aliskiren increases bradykinin and tissue kallikrein mRNA levels in the heart

1. Aliskiren is a renin inhibitor with an IC₅₀ of 0.6 nmol/L for human renin, 4.5 nmol/L for mouse renin and 80 nmol/L for rat renin. 2. In the present study, we compared the effects of aliskiren (10 mg/kg per day), the angiotensin‐converting enzyme inhibitor perindopril (0.2 mg/kg per day) and their combination on angiotensin and bradykinin peptides in female heterozygous (mRen‐2)27 rats, transgenic for the mouse renin gene. 3. All three treatments produced similar reductions in systolic blood pressure, heart weight and plasma aldosterone levels and reduced angiotensin II levels in lung, but only perindopril and the combination reduced angiotensin II levels in kidney of (mRen‐2)27 rats. In contrast, aliskiren and the combination, but not perindopril alone, increased cardiac bradykinin levels. Aliskiren increased immunostaining for tissue kallikrein in the heart and reduced cardiac fibrosis. 4. We investigated the mechanism underlying the increase in bradykinin levels following aliskiren treatment in Sprague–Dawley rats, in which aliskiren has a lower potency for renin inhibition. Aliskiren (10 mg/kg per day) reduced renal angiotensin levels within 24 h, but treatment for > 24 h was required to increase cardiac bradykinin levels. Moreover, 3 mg/kg per day aliskiren increased cardiac bradykinin levels, but did not reduce renal angiotensin levels. Aliskiren did not potentiate the hypotensive effects of bradykinin; however, it increased tissue kallikrein, but not plasma kallikrein, mRNA levels in the heart. 5. These data demonstrate that the aliskiren‐induced increase in cardiac bradykinin levels is independent of renin inhibition and changes in bradykinin metabolism, but is associated with increased tissue kallikrein gene expression.     

EFFECT OF LONG-TERM TREATMENT WITH AN ANGIOTENSIN-CONVERTING ENZYME INHIBITOR ON THE RENIN-ANGIOTENSIN SYSTEM IN SPONTANEOUSLY HYPERTENSIVE RATS

1. To obtain information on regulation of the brain renin-angiotensin system, the effect of long-term administration of angiotensin-converting enzyme (ACE) inhibitor on brain renin and angiotensinogen mRNA was studied. 2. Spirapril (3 mg/kg) was orally administered daily for 8 weeks to spontaneously hypertensive rats (SHR) from 12 weeks after birth. Renin and angiotensinogen mRNA in the brain and kidney were then quantitated by Northern blot analyses with [32P]-labelled rat renin and angiotensinogen cDNA as hybridization probes. Plasma renin activity (PRA), angiotensin II (AII) concentration, plasma ACE activity and brain tissue ACE activity were also measured. 3. Compared with the control group, the Spirapril-treated group had significantly lower blood pressure (P less than 0.01), significantly higher PRA (P less than 0.01), a not significantly different plasma AII concentration, and lower plasma and brain ACE activities (P less than 0.01). Interestingly, the brain renin and angiotensinogen mRNA levels of the two groups were similar, but the renal renin mRNA level was significantly higher in the Spirapril-treated group (P less than 0.01). 4. These results indicate that the mRNA levels of brain renin and angiotensinogen were not affected by chronic ACE inhibition in the circulation and suggest that AII in the brain might not be affected by systemic ACE inhibition.     

RAD51基因在结直肠癌中的表达及与预后的关系

目的 探讨RAD51基因在结直肠癌（CRC）中的表达及其与临床病理特征和预后的关系。方法 收集72例CRC患者的病理标本（CRC组,n=72）,以及经病理证实无癌细胞存在、距肿瘤边缘>3 cm的肠黏膜组织（癌旁对照组,n=72）。采用qRT-PCR和Western blotting技术对CRC与癌旁组织的RAD51 mRNA和蛋白表达水平进行检测。影响CRC患者预后的因素采用Cox比例风险模型分析。预后分析采用Kaplan-Meier和Log-rank法。结果 CRC组RAD51 mRNA和蛋白相对表达量均高于癌旁对照组,差异有统计学意义（P<0.05）。CRC组织中RAD51表达水平与TNM分期有关（P<0.05）。RAD51高表达是影响CRC患者5年无瘤生存率和5年总体生存率的危险因素（P<0.05）。RAD51低表达的CRC患者5年总体生存率和5年无瘤生存率均高于RAD51高表达患者（P<0.05）。结论 RAD51在CRC组织中高表达,与TNM分期有关。RAD51高表达的CRC患者预后差,RAD51可能是临床上预测CRC患者预后的一个潜在指标。     

CDCA8通过调控增殖促进前列腺癌发生的作用机制研究

目的 探讨细胞分裂周期相关蛋白8（CDCA8）在前列腺癌（PCa）中的表达及作用机制。方法 通过生物信息学方法分析正常前列腺组织和PCa组织中CDCA8 mRNA水平差异。利用癌症基因组图谱（TCGA）数据库中RNA表达测序数据分析CDCA8表达相关的PCa患者无病生存期（DFS）。免疫组织化学方法检测根治性前列腺切除...     

17beta-estradiol inhibits angiotensin II-induced collagen synthesis of cultured rat cardiac fibroblasts via modulating angiotensin II receptors

Circulating endogenous estrogen is considered to be cardiovascular protective, but the underlying mechanisms remain obscure. The cardiac fibroblasts, the most abundant cell type present in the heart, are responsible for the deposition of extracellular matrix. Angiotensin II has been known to stimulate cardiac collagen gene expression. The present study was designed to investigate the effect of 17beta-estradiol on the angiotensin II-induced proliferation and collagen synthesis in cultured cardiac fibroblasts by using real-time polymerase chain reaction (PCR), Western blot and 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide proliferation assay. Angiotensin II increased the cell proliferation and synthesis of collagen types I and III. Angiotensin II up-regulated the gene expression of the angiotensin AT(1) receptor and down-regulated the gene expression of the angiotensin AT(2) receptor in cardiac fibroblasts. The effects of angiotensin II was abolished by the angiotensin AT(1) receptor antagonist, losartan, but not by the angiotensin AT(2) receptor antagonist, PD 123319. 17beta-estradiol prevented increases in proliferation and attenuated the collagen synthesis in response to angiotensin II. The increased AT(1) receptor mRNA levels and decreased AT(2) receptor mRNA levels were partially reversed by 17beta-estradiol treatment. In conclusion, the down-regulation of angiotensin AT(1) receptor expression and function is likely to be an important mechanism accounting for the inhibitory effect of 17beta-estradiol on angiotensin II-stimulated proliferation and collagen synthesis in cardiac fibroblasts. This effect may confer at least in part the cardiac protective action of 17beta-estradiol under pathological conditions with increased activity of the rennin-angiotensin system.     

Chymase mediates paraquat-induced collagen production in human lung fibroblasts

Survivors of paraquat poisoning may be left with pulmonary fibrosis and a restrictive type of pulmonary dysfunction. Chymase converts angiotensin (Ang) I to Ang II, which is closely involved with lung fibrosis. The role played by chymase in paraquat-induced lung fibrosis is unclear. We examined the effects of paraquat on chymase, renin–angiotensin system components, and collagen expression in murine and human lung fibroblasts (MRC-5). Lung chymase and collagen type I mRNA and protein expression were significantly increased and angiotensin-converting enzyme (ACE) mRNA and protein expression were comparable between the control and paraquat-treated mice 1 and 3 weeks after administration. Paraquat significantly upregulated angiotensinogen mRNA expression in a dose-dependent manner while ACE activity and protein expression were similar in MRC-5 cells. Furthermore, paraquat enhanced Ang II and collagen type I mRNA and protein expression, α-smooth muscle actin, and chymase protein and chymase small interfering RNA inhibited these effects. The cDNA sequence of chymase in MRC-5 cells is identical to that in human mast cells. This study found increased chymase expression in paraquat-treated human lung fibroblasts and confirmed in vitro and in an in vivo paraquat model of lung fibrosis that chymase generates Ang II and enhances collagen expression. These data suggest a role for chymase in the pathogenesis of paraquat-induced lung fibrosis.     

基于数据挖掘分析GPR87在非小细胞肺癌中的表达及临床意义

目的 探索G蛋白偶联受体87（GPR87）在非小细胞肺癌（NSCLC）中的表达及意义。方法 通过GE-IA在线工具分析TCGA数据库中GPR87 mRNA在NSCLC和正常肺组织中的表达情况,并进一步分析GPR87基因表达与NSCLC患者TNM分期和总生存期（OS）之间的关系。利用String在线工具分析与GPR87相互作用的蛋白。结果 GPR87 mRNA在肺腺癌（LUAD）和肺鳞癌（LUSC）组织中的表达水平均明显高于正常肺组织（P<0.01）。GPR87 mRNA高表达倾向于与NSCLC患者临床进展期有关,但差异无统计学意义（P>0.05）。TCGA数据库中,GPR87 mRNA高表达组LUAD患者的OS明显短于低表达组（P=0.006 5）,并在Kaplan-Meier Plotter数据库中得到了进一步验证（P=0.029）。然而,在TCGA数据库中,GPR87 mRNA高表达组LUSC患者的OS明显长于低表达组（P=0.036）,但在Kaplan-Meier Plotter数据库中差异无统计学意义（P=0.35）。String分析显示,LPAR1、LPAR2、LPAR3、SCIN、OR56A3、OR52W1、OR52L1、OR56B4、OR56A1、OR52B2等蛋白与GPR87有明显的相互作用。结论 基于肿瘤基因数据库信息挖掘,GPR87在NSCLC组织中呈高表达,并与LUAD患者预后不良相关,可能是LUAD的潜在分子标志物。     

Effects of olmesartan, an AT1 receptor antagonist, on hypoxia-induced activation of ERK1/2 and pro-inflammatory signals in the mouse lung

The present study aimed to investigate the effects of olmesartan, an antagonist for angiotensin II receptor type 1(AT1), on the activation of extracellular signal-regulated kinases (ERK)1/2, tissue remodeling, and pro-inflammatory signals in the right ventricle and lung of mice during the early phase of hypobaric hypoxia. Phosphorylation of ERK1/2 in both tissue types in response to hypoxia peaked at 1–3 days, and declined rapidly in the right ventricle, whereas in the lung it was sustained for at least 8 days. Upregulation of angiotensinogen mRNA was observed in the hypoxic lung at 4–9 days, but not in the hypoxic right ventricle and pulmonary artery. Olmesartan inhibited the hypoxia-induced phosphorylation of ERK1/2 in the lung, but not in the right ventricle. Neither right ventricular hypertrophy nor the thickening of the intrapulmonary arterial wall was ameliorated by olmesartan. However, this drug inhibited the expression of the mRNA for angiotensinogen and several pro-inflammatory factors, including interleukin-6 and inducible nitric oxide synthase in the hypoxic lung. These results suggest that olmesartan blocks a potential positive feedback loop of the angiotensin II-AT1 receptor system, which may lead to attenuate pro-inflammatory signals in the mouse lung, that are associated with hypoxic pulmonary hypertension, without inducing any appreciable effects on the compensatory cardiopulmonary hypertrophy at an early phase of exposure to a hypobaric hypoxic environment.     

Proceedings of the Symposium ‘Angiotensin AT1 Receptors: From Molecular Physiology to Therapeutics’: TRANSGENIC RATS: TOOLS TO STUDY THE FUNCTION OF THE RENIN-ANGIOTENSIN SYSTEM

• 1 The development of the transgenic technology for the rat allowed the evaluation of gene functions in the cardiovascular system in vivo. New insights have been gained particularly in the functions of the renin-angiotensin system (RAS), as most transgenic rat models established so far carry genes of this system.
• 2 TGR(mREN2)27 is a rat harbouring the mouse Ren-2 gene and exhibiting fulminant hypertension. The plasma RAS in this animal is down-regulated; however, the tissue-specific production of angiotensin II is activated (e.g. in the adrenal gland, the brain and the vessel wall). The physiological consequences of this activation, which finally leads to hypertension, can be studied in TGR(mREN2)27, rendering it a valuable tool in the functional analysis of tissue RAS.
• 3 TGR(hREN) and TGR(hAOGEN) carry the human genes for renin and angiotensinogen, respectively. In these animals the species-specific interaction of the two proteins and the expression pattern of the genes can be studied. Furthermore, these animals can be used to test renin-inhibitory drugs for use in antihypertensive therapy.
• 4 Further refinement of transgenic methodology (e.g. by the development of gene targeting in rats), should enhance our understanding of the functions of the RAS in cardiovascular regulation.