Autophagy in vascular endothelial cells

The importance of autophagy in cardiovascular physiology and cardiovascular disease is increasingly recognized; however, the precise biological effects and underlying mechanisms of autophagy in the cardiovascular system are still poorly understood. In the last few years, the effects of autophagy in endothelial cells have attracted great interests. This article provides a summary of our current knowledge on the regulatory factors, signalling mechanisms, and functional outcomes of autophagy in endothelial cells. It is suggested that in most situations, induction of an autophagic response has cytoprotective effects. The beneficial effects of autophagy in endothelial cells are likely to be context‐dependent, since autophagy may also contribute to cell death under certain circumstances. In addition to regulating endothelial cell survival or death, autophagy is also involved in modulating other important functions, such as nitric oxide production, angiogenesis and haemostasis/thrombosis. The mounting data will help us draw a clear picture of the roles of autophagy in endothelial cell biology and dysfunction. Given the pivotal role of endothelial dysfunction in the pathogenesis of vascular disease, disruptions of autophagy in endothelial cells are likely to have significant contributions. This is supported by some preliminary ex vivo data indicating that compromised autophagic functions may be important in the development of endothelial dysfunctions associated with diabetes and ageing.     

Diabetes-induced enhancement of prostanoid-stimulated contraction in mesenteric veins of mice

We investigated the influence of the diabetic state on the contractile response of longitudinal segments of isolated mesenteric vein to prostanoids and leukotriene (LT), and the contribution of the vascular endothelium to modulation of the contractile response was determined. The normal mesenteric vein and de-endothelialized veins of normal (ddY), diabetic KK-CAy and streptozotocin ddY mice (150 mg/kg, i.v., 6 weeks) were used. In the diabetic state, the contractions produced by noradrenaline (60 microM), high K+ solution (143.4 mM), and the thromboxane A2 analogue U-46619 (29 nM-29 mM) were not affected, and LTD4 (0.1 nM-1 microM)-induced contraction was suppressed. Contractions induced by prostaglandin (PG) E2 (0.2 microM-2 mM), PGF2 alpha (0.3 microM-0.3 mM) and the prostacyclin derivatives PGI2-Na (10-100 microM) and TRK-100 (0.2 microM-2 mM) were significantly enhanced in the presence of an intact vascular endothelium, but not in de-endothelialized segments. The increase in PGF2 alpha (0.28 mM) contractions was dependent on age (correlation coefficient r = 0.36, significant difference, P less than 0.05) and blood glucose (r = 0.88, significant difference, P less than 0.01), but was independent of obesity. The contractile response to PGD2 (0.3-0.9 mM) was enhanced in both intact and de-endothelialized segments. These results indicate that the diabetic state affects prostanoid responses in an endothelium-dependent manner, except for the PGD2 response, which is independent of the endothelium.     

糖尿病大鼠甲状腺组织的超微病理改变

目的：探讨糖尿病大鼠甲状腺组织超微结构的变化，了解甲状腺组织是否亦为糖尿病慢性攻击的靶器官。方法：Wistar大鼠60只，随机分为糖尿病模型组（n=48）和正常对照组（n=12）。经四氧嘧啶造模8周后处死，取甲状腺组织经固定、乙醇脱水、Epon812包埋、超薄切片、醋酸铀和柠檬酸铅染色后，日本电子100-CX透射电镜进行观察。结果：糖尿病大鼠甲状腺电镜下无一例为正常甲状腺组织结构。大多数显示甲状腺滤泡上皮细胞微绒毛膜大量减少，有的几乎全部消失；滤泡上皮细胞核变得小而扁平、内陷；粗面内质网扩张，池内有明显增多的髓磷体；线粒体、溶酶体数目减少功能低下。部分组织可见甲状腺旁细胞增生现象，细胞核大，核染色质活跃，胞浆内神经分泌颗粒有的丰富有的减少。结论：甲状腺组织亦为糖尿病攻击的靶器官，组织形态变化显示甲状腺功能低下，甲状腺旁细胞功能活跃。     

Lamin A/C在干细胞表观遗传药理学中的调控作用

Lamin A/C是核纤层的主要组成部分,在干细胞表观遗传调控中的重要作用近年来越来越被认识。Lamin A/C不仅可以起到维持核内稳态和保护染色质的作用,同时也可以作为机械刺激细胞核内的反应器,将力学信号传导至核内,可直接或间接参与基因的转录调控。该文主要综述微环境中力学信号对干细胞Lamin A/C的调节,以及Lamin A/C的功能变化对染色体修饰、基因表达调控的影响。     

The epigenetic face of lupus: Focus on antigen-presenting cells

In recent years, epigenetic mechanisms became widely known due to their ability to regulate and maintain physiological processes such as cell growth, development, differentiation and genomic stability. When dysregulated, epigenetic mechanisms, may introduce gene expression changes and disturbance in immune homeostasis leading to autoimmune diseases. Systemic lupus erythematosus (SLE), the most extensively studied autoimmune disorder, has already been correlated with epigenetic modifications, especially in T cells. Since these cell rely on antigen presentation, it may be assumed that erroneous activity of antigen-presenting cells (APCs), culminates in T cell abnormalities. In this review we summarize and discuss the epigenetic modifications in SLE affected APCs, with the focus on dendritic cells (DCs), B cells and monocytes. Unravelling this aspect of SLE pathogenesis, might result in identification of new disease biomarkers and putative therapeutic approaches.     

Diabetes-induced changes in 5-hydroxytryptamine modulation of vagally-induced bradycardia in rat heart

1. In the present study, we investigated how alloxan-induced diabetes affects the ability of 5-hydroxytryptamine (5-HT) to modulate bradycardia induced in vivo by electrical stimulation of the vagus nerve in pithed rats. We also analysed the type and/or subtype of 5-HT receptors involved. 2. Diabetes was induced in male Wistar rats with a single injection of alloxan (150 mg/kg, s.c.). Four weeks later, rats were anaesthetized, pretreated with atenolol and pithed. Electrical stimulation (3, 6 and 9 Hz) of the vagus nerve resulted in frequency dependent decreases in heart rate (HR). 3. In diabetic rats, intravenous bolus administration of high doses of 5-HT (100 and 200 microg/kg) increased the bradycardia induced by vagal electrical stimulation. Similarly, low doses (10 microg/kg) of the 5-HT(1/7) receptor agonist 5-carboxamidotryptamine (5-CT), increased vagally induced bradycardia. However, at high doses (50, 100 and 150 microg/kg), 5-CT reduced the bradycardia. Attenuation of the vagally induced bradycardia evoked by the higher doses of 5-CT was reproduced by L-694,247 (50 microg/kg), a selective agonist for the non-rodent 5-HT(1B) and 5-HT(1D) receptors. Enhancement of the vagally induced bradycardia elicited by low doses of 5-CT was reproduced by the selective 5-HT(1A) receptor agonist 8-hydroxydipropylaminotretalin hydrobromide (8-OH-DPAT; 50 microg/kg). These stimulatory and inhibitory actions on vagal stimulation-induced bradycardia in diabetic rats were also observed after administration of exogenous acetylcholine. 4. Vagally induced bradycardia in diabetic rats was not affected by administration of the selective 5-HT(2) receptor agonist alpha-methyl-5-HT (150 microg/kg), the selective 5-HT(3) receptor agonist 1-phenylbiguanide (150 microg/kg) or the selective 5-HT(1B) receptor agonist CGS-12066B (50 microg/kg). 5. Enhancement of the electrical stimulation-induced bradycardia in diabetic rats caused by 5-CT (10 microg/kg) or 8-OH-DPAT (50 microg/kg) was abolished by the selective 5-HT(2/7) receptor antagonist mesulergine (1 mg/kg) and the selective 5-HT(1A) receptor antagonist WAY-100,635 (100 microg/kg), respectively. Similarly, pretreatment with the non-selective 5-HT(1) receptor antagonist methiothepin (0.1 mg/kg) blocked the inhibitory effect of 5-CT (50 microg/kg) on the bradycardia induced by vagal electrical stimulation in diabetic rats. BRL-15572 (2 microg/kg), a selective 5-HT(1D) receptor antagonist, inhibited the action of L-694,247 (50 microg/kg), a selective agonist for the non-rodent 5-HT(1B) and 5-HT(1D) receptors, on the vagally induced bradycardia. 6. In conclusion, in the present study, experimental diabetes evoked changes in both the nature and 5-HT receptor types/subtypes involved in vagally induced bradycardia.     

血管内皮生长因子受体-3在血管内皮细胞和平滑肌细胞中的表达

目的探讨血管内皮细胞生长因子受体-3（VEGFR-3）在血管内皮细胞和平滑肌细胞中的表达。方法分离人脐带中的脐静脉和脐动脉,冰冻切片后以免疫荧光法检测VEGFR-3的表达。结果VEGFR-3荧光信号表达于脐静脉内皮细胞和平滑肌细胞,以及脐动脉内皮细胞、平滑肌细胞和外周细胞。结论血管内皮细胞和平滑肌细胞均表达VEGFR-3。     

Embryonic signature in breast cancers; Pluripotency roots of cancer stem cells

Drug discovery programs for preclinical oncology typically select compounds which have a predilection for inducing cytotoxic effects in cancer cell lines and subsequently, for inhibiting the growth of the transplanted cancer cells in vivo (Winquist et al., 2010). Unfortunately, the cytotoxic effect in vitro and inhibition of tumor growth in animal models are not the end story for curing cancer in preclinical models. The reason behind that is the exciting of small sub type of cells that are relatively resistance to therapy and able to repopulate in vivo, called cancer stem cells (CSCs). O leis et al. recently reported that the pluripotency gene Sox2 but not Oct4 or Nanog is expressed in early stage of breast tumor. Furthermore, the authors demonstrated that Sox2 downregulation, inhibited mammosphere formation and delayed tumor formation in xenograft tumor initiation models (Leis et al., 2012). In this review, we will shed the light on the importance of Sox2 in breast and other tissue tumorigenesis and associated aggressiveness.     

静脉血管平滑肌细胞中的多倍体细胞分析

目的探讨流式细胞术分析静脉血管平滑肌细胞多倍体细胞的方法和意义。方法选取普通级健康成年日本大耳白兔2只,雄性,8月龄,大约3 kg;普通级SD大鼠2只,雄性,8周龄,大约200 g。人大隐静脉为2例心脏冠状动脉旁路移植术中修补血管所得。在麻醉日本大耳白兔和大鼠后开腹腔获取下腔静脉,采用多种酶组合的消化液消化兔、大鼠以及人的静脉血管得到适合流式细胞检测的静脉血管平滑肌单细胞悬液,用碘化丙啶标记细胞,利用细胞流式仪分析兔、大鼠以及人的静脉血管平滑肌单细胞悬液各5 000个细胞,检测细胞DNA含量,DNA含量翻倍的细胞为多倍体细胞。并利用荧光原位杂交技术（FISH）验证阳性对照HEK293细胞系中的多倍体的存在。结果流式结果显示分析的5 000个细胞中,阳性对照HEK293细胞的多倍体细胞为1 355个（27.1%）,从2例患者中取得的大隐静脉多倍体细胞为310个（6.2%）和250个（5.0%）;2只大鼠的下腔静脉平滑肌细胞中多倍体细胞为360个（7.2%）和450个（9%）;2只兔的下腔静脉平滑肌细胞中多倍体细胞分别为270个（5.4%）和305个（6.1%）。结论成年的静脉血管包括人大隐静脉的平滑肌细胞中普遍存在一定比例的多倍体细胞。     

ATP release pathways in vascular endothelial cells

Vascular endothelial cells regulate vascular tonus, growth, and angiogenesis in response to mechanical stresses. ATP release is one of well-known mechanosensitive responses in endothelial cells. Released ATP induces Ca(2+) responses and nitric oxide production in neighboring cells in an auto/paracrine manner. Mechanosensitive and agonist-induced ATP releases are also observed in other cell types, but the cellular mechanisms and pathways of ATP release are largely unknown. Reported candidates for ATP release pathways are ABC proteins including P-glycoprotein and CFTR, exocytosis of ATP-containing vesicles, and ATP-permeable anion channels. In vascular endothelium, vesicular exocytosis, volume-regulated anion channels (VRAC), and connexin hemichannels have been reported as candidates for ATP release pathways. We found that VRAC inhibitors suppressed hypotonic stress-induced ATP release in bovine aortic endothelial cells. Furthermore, extracellular ATP suppressed VRAC current in a voltage dependent manner, which could be fitted to the permeation-blocker model with a Kd(0) of 1 mM and delta value of 0.41. However, it should be noted that VRAC is probably not the only pathway for ATP release in the endothelium, because basal ATP release was not inhibited by VRAC inhibitors. Further investigations are definitely warranted to clarify the details and therapeutic significance of mechanosensitive ATP release in the endothelium.     

Diabetes-induced changes in endothelial mechanisms implicated in rabbit carotid arterial response to 5-hydroxytryptamine

The influence of diabetes on endothelial mechanisms implicated in the response of isolated rabbit carotid arteries to 5-hydroxytryptamine (5-HT) was studied. 5-HT induced a concentration-dependent contraction that was potentiated in arteries from diabetic rabbits with respect to that in arteries from control rabbits. Endothelium removal potentiated 5-HT contractions in arteries from both control and diabetic rabbits but increased the maximum effect only in arteries from diabetic rabbits. Incubation of arterial segments with N(G)-nitro-L-arginine (L-NA) enhanced the contractile response to 5-HT. This L-NA enhancement was greater in arteries from diabetic rabbits than in arteries from control rabbits. Aminoguanidine did not modify the 5-HT contraction in arteries from control and diabetic rabbits. Indomethacin inhibited the 5-HT-induced response, and this inhibition was higher in arteries from control rabbits than in arteries from diabetic rabbits. In summary, diabetes enhances the sensitivity of the rabbit carotid artery to 5-HT. In control animals, the endothelium modulated the arterial response to 5-HT by the release of both nitric oxide (NO) and a vasoconstrictor prostanoid. Diabetes enhances endothelial constitutive NO activity and impairs the production of the endothelial vasoconstrictor.     

线粒体融合蛋白2在原发性高血压患者血管组织和平滑肌细胞中表达的研究

目的 观察线粒体融合蛋白2(Mfn2)在人血管组织和平滑肌细胞(VSMCs)中的表达情况.方法 选取2006年10月至2007年10月在北京安贞医院行冠状动脉旁路移植术的患者60例,分为高血压组和正常血压组各30例,收集术中剩余桥血管(乳内动脉).取1例正常产妇产新生婴儿脐带,进行人乳内动脉和VSMCs培养.提取乳内动脉组织和VSMCs总RNA,RNA的反转录-cDNA的聚合酶链式扩增(RTPCR),观察2组患者Mfn2的表达情况.用不同浓度血小板源性生长因子(PDGF-BB)诱导VSMCs增殖并检测Mfn2表达情况.结果 2组患者血压水平、体质指数差异有统计学意义(P＜0.05).人乳内动脉血管组织和VSMCs均有Mfn2表达,高血压组Mfn2表达水平均低于正常血压组(人乳内动脉血管组织:0.42±0.10比0.49 ±0.12,P=0.014;VSMCs:0.29 ±0.07比0.33 ±0.08,P=0.041).高血压组原代VSMCs细胞计数高于正常血压组(0.48 ±0.15比0.38 ±0.11,P=0.005).与对照组相比,随PDGF-BB浓度增加VSMCs计数增加(对照组:0.36 ±0.09,10 μg/L组:0.53 ±0.13,100 μg/L组:0.54±0.12),VSMCs的Mfn2表达减少(对照组:0.31 ±0.08,10μg/L组:0.22±0.06,100μg/L组:0.21±0.05,P＜0.05);100 μg/L组与10 μg/L组相比,VSMCs细胞计数和VSMCs的Mfn2表达差异无统计学意义(P值分别为0.862和0.755).结论 在人血管组织和培养的VSMCs中验证了高血压和正常血压入选者存在Mfn2的表达差别.     

Cytotoxic effects produced by arecoline correlated to epigenetic regulation in human K-562 cells

Epigenetic effects are considered heritable but may also be modified by environmental factors. Arecoline (ARC), a major component of areca nut alkaloids, is an important environmental risk factor for oral cancer and hepatocellular carcinomain Taiwan. The aim of this study was to determine the influence of ARC on the epigenome. The mRNA expression of histone methyltransferases, acetyltransferases, and demethylases were assessed in K-562 cells following exposure to ARC. Results demonstrated that ARC produced changes in the expressions of several genes catalyzing histone methylation (Mll, Setdb1, and Suv39h2), acetylation (Atf2), and demethylation (JMJD6). Since H3K9 methylation is involved in maintaining the stability of heterochromatin structures and inactivating euchromatic gene expressions, data suggest that the ARC-induced epigenetic changes play a role in the mechanisms underlying chemical-mediated cytotoxicity and genotoxicity.     

Levamisole induced apoptosis in cultured vascular endothelial cells

To better understand the anticancer activity of Levamisole (LMS), which serves as an adjuvant in colon cancer therapy in combination with 5-Fluorouracil, this study analyses LMS' ability to induce apoptosis and growth arrest in cultured human micro- and macrovascular endothelial cells (ECs) and fibroblasts. Cells exposed (24 h) to Levamisole (range: 0.5 - 2 mmol l(-1)) alone or in combination with antioxidants (10 mmol l(-1) glutathione or 5 mmol l(-1) N-Acetylcysteine or 0.1 mmol l(-1) Tocopherol) were evaluated for apoptosis ((3)H-thymidine assays, in situ staining), mRNA/protein expression (Northern/Western blot), and proliferation ((3)H-thymidine incorporation). Levamisole dose-dependently increased apoptosis in ECs to 230% (HUVECs-human umbilical vein ECs), 525% (adult human venous ECs) and 600% (human uterine microvascular ECs) but not in fibroblasts compared to control cells (set as 100%). Levamisole increased in ECs integrin-dependent matrix adhesion, inhibited proliferation (-70%), reduced expression of survival factors such as clusterin (-30%), endothelin-1 (-43%), bcl-2 (-34%), endothelial NO-synthase (-32%) and pRb (Retinoblastoma protein: -89%), and increased that of growth arrest/death signals such as p21 (+73%) and bak (+50%). LMS (2 mmol l(-1))-induced apoptosis was inhibited by glutathione (-50%) and N-Acetylcysteine (-36%), which also counteracted reduction by Levamisole of pRb expression, suggesting reactive oxygen species and pRb play a role in these processes. The ability of LMS to selectively induce apoptosis and growth arrest in endothelial cells potentially hints at vascular targeting to contribute to Levamisole's anticancer activity.     

Methylglyoxal-induced mitochondrial dysfunction in vascular smooth muscle cells

The effects of methylglyoxal (MG) on mitochondria with specific foci on peroxynitrite (ONOO⁻) production, manganese superoxide dismutase (MnSOD) activity, and mitochondrial functions in vascular smooth muscle A-10 cells were investigated. Mitochondrial MG content was significantly increased after A-10 cells were treated with exogenous MG, and so did advanced glycated endproducts (AGEs) formation, indicated by the appearance of N_?-(carboxyethyl) lysine, in A-10 cells. The levels of mitochondrial reactive oxygen species (mtROS) and ONOO⁻ were significantly increased by MG treatment. Application of ONOO⁻ specific scavenger uric acid lowered the level of mtROS. MG significantly enhanced the production of mitochondrial superoxide (O₂⁻) and nitric oxide (NO), which were inhibited by SOD mimic 4-hydroxy-tempo and mitochondrial nitric oxide synthase (mtNOS) specific inhibitor 7-nitroindazole, respectively. The activity of MnSOD was decreased by MG treatment. Furthermore, MG decreased respiratory complex III activity and ATP synthesis in mitochondria, indicating an impaired mitochondrial respiratory chain. AGEs cross-link breaker alagebrium reversed all aforementioned mitochondrial effects of MG. Our data demonstrated that mitochondrial function is under the control of MG. By inhibiting Complex III activity, MG induces mitochondrial oxidative stress and reduces ATP production. These discoveries will help unmask molecular mechanisms for various MG-induced mitochondrial dysfunction-related cellular disorders.