Adhesion and Function of Human Endothelial Cells Co-cultured on Smooth Muscle Cells

To evaluate interactions between human endothelial cells (ECs) and smooth muscle cells (SMCs) for the development of tissue-engineered vessels, we examined the adhesion and key cell properties of human ECs grown on quiescent human aortic SMCs. ECs attached to SMCs spread more slowly than ECs attached to fibronectin surfaces, and ECs aligned along the direction of the SMCs. ECs attached firmly and less than 5% of the cells were removed by shear stresses as high as 300 dyn cm⁻². Unlike porcine SMCs and co-cultures, human SMCs or co-cultures do not contract under flow, and the human ECs and SMCs in co-culture align toward the direction of flow. A confluent endothelium could be maintained in co-culture for over 30 days, and some of the ECs reoriented perpendicular to the SMCs after 9 days in static culture. Surface tissue factor levels in ECs and SMCs were less in co-culture than in monoculture. Co-culture induced an increase in calponin expression in SMCs. These findings show that human co-cultures can be maintained for long culture periods, where the endothelium remains confluent and responds to long-term exposure to flow, and EC–SMC interactions lead to an increase in SMC differentiation and an EC surface that is less thrombotic.     

Aortic Smooth Muscle Cells Express and Secrete Vascular Endothelial Growth Factor

Abstract

We examined whether cultured bovine aortic smooth muscle (ASM) cells express VEGF. RNA blot analysis of total cellular RNA derived from ASM cells demonstrates the expression of the VEGF gene. ASM cells release in the medium a VEGF-like endothelial cell mitogen which binds to heparin-sepharose and has an apparent molecular weight of 40-45 kDa as assessed by an HPLC gel filtration column. Consistent with VEGF, this mitogen does not stimulate the proliferation of ASM cells. Immunoblot analysis of the bioactive material with an antibody specific for VEGF demonstrates the presence of a major immunoreactive band with an apparent molecular mass of 23 kDa and a minor band with a molecular mass of -18 kDa, in reducing conditions. The major band has very similar apparent molecular weight as the 165 amino-acid species of human recombinant VEGF of folliculo-stellate cells derived VEGF. These data demonstrate the expression and synthesis of VEGF by cultured ASM cells and suggest that the 164 amino-acid species is the predominant molecular form of the growth factor secreted by such cells. VEGF released by ASM cells may play a paracrine role in the maintenance of the integrity of the endothelial lining or in the abnormal proliferation of the vasa vasorum which takes place in atherosclerosis.     

巨噬细胞增强H2O2抑制人血管内皮细胞和兔血管平滑肌细胞的增殖

目的探讨H2O2处理对巨噬细胞分泌因子的影响,及对血管内皮细胞(VEC)及平滑肌细胞(VSMC)增殖的影响。方法实验分组:空白对照组,0.5 mmol/L H2O2组,巨噬细胞条件培养基组,巨噬细胞+0.5 mmol/L H2O2条件培养基组。CCK-8测定VEC和VSMC的增殖情况。ELISA检测白介素-6(IL-6)、白介素-10(IL-10)、单核细胞趋化蛋白-1(MCP-1)、肿瘤坏死因子-α(TNF-α),以及血管内皮细胞生长因子(VEGF)。Western blot检测增殖核抗原(PCNA)、细胞周期蛋白cyclinD1的表达,以及血管内皮细胞内皮源性一氧化氮合酶(eNOS)的表达。结果 VEC中其他3个处理组的增殖水平均低于对照组(P<0.05),而在VSMC中,巨噬细胞组的增殖水平高于空白对照组(P<0.05)。在VEC和VSMC中,巨噬细胞+0.5 mmol/L H2O2组的增殖水平均高于单独0.5 mmol/L H2O2处理组(P<0.05),且低于单独巨噬细胞处理组(P<0.05)。巨噬细胞分泌的IL-6、IL-10、MCP-1随时间和H2O2的浓度增加而增高(P<0.05)。VEC分泌的VEGF在1.0 mmol/L H2O2浓度时有明显增高(P<0.05)。结论氧化应激处理可影响巨噬细胞炎性因子的分泌,进而影响血管内皮细胞和平滑肌细胞的增殖。     

Soluble Cytostatic Factor(s) Released from Human Monocytes

Human monocytes release a stable cytostatic factor during in vitro culture after stimulation with lymphokine and endotoxin. The cell cycle time of synchronized NHTK 3025 cells increased from 20.3 to 23.2 h in the first cell cycle when the target cells were exposed to the factor during the whole cell cycle. In exponentially growing NHIK 3025 cell cultures the cell doubling time increased from 18.9 h to 23. l h under continuous factor exposure for 70 h. These cells regained normal cell division rate when fresh culture medium replaced the cytostatic factor. Continuous exposure to the cytostatic factor for 96 h increased the cell doubling time of asynchronous K-562 cells from 19.7 to 31.8 h. The target cell DNA synthesis, evaluated by thymidine incorporation. was markedly depressed after culture with the factor for 24 h, and this depression was detectable already within 4 h of culture. The factor showed no cytolytic effect on the two cell lines tested. The cytostatic factor influenced the cell cycle distribution of both target cells tested, since cell cycle analysis by DNA flow cytometry demonstrated a reversible inhibition of factor-exposed NHIK 3025 cells in G₁- and early S-phase, whereas K-562 cells accumulated in G₁-phase.     

Soluble Cytostatic Factor(s) Released from Human Monocytes

Human monocytes activated with lymphokines after 3–7 days of in vitro maturation released stable cytostalic factor(s) (CF), as determined by reduction of [methyl-³H]thymidine incorporation and cell counts in (arget cell cultures, Lipopolysaccharide from Escherichia coli slightly enhanced the release of CF. The CF release was greatest at an intermediate stage of the in vitro monocyte maturation to large, macrophage-like cells. Three transformed human cell lines (NHIK 3025, K-562, and U-20S) and normal skin fibroblasts were consistently inhibited by CF. Normal human mesothelial cells were variably affected, while normal human lymphocytes proliferating in response to allogeneic cells in mixed lymphocyte culture were not inhibited. Further analysis of CF may disclose one of the mechanisms whereby human monocytes and macrophages inhibit growth of human tumour cells.     

大鼠提睾肌缺血再灌注损伤时血管平滑肌细胞和内皮细胞的增殖与凋亡

目的研究大鼠提睾肌缺血再灌注后血管平滑肌细胞和内皮细胞的增殖和凋亡。方法以免疫组织化学SP法和末端脱氧核苷酸转移酶介导生物素标记法 (TUNEL)对大鼠提睾肌缺血再灌注后的血管平滑肌细胞和内皮细胞的Bcl-2、Bax、P53和增殖细胞核抗原 (PCNA)的表达进行观察研究。结果缺血再灌注后 ,血管平滑肌细胞和内皮细胞均有Bcl-2、Bax、P53和PCNA的表达 ,在平滑肌细胞 ,Bcl-2阳性细胞数量明显高于Bax和P53。PCNA阳性细胞明显增多。在内皮细胞 ,Bax和P53的表达最强 ,TUNEL阳性细胞率最高。结论大鼠提睾肌缺血再灌注可造成平滑细胞的增殖和内皮细胞的凋亡 ,其结果可能与微循环障碍有关。     

Contractile Proteins of Endothelial Cells, Platelets and Smooth Muscle

In experiments described herein it was observed, by direct and indirect immunofluorescence technics, that rabbit antisera to human platelet actomyosin (thrombosthenin) stained mature megakaryocytes, blood platelets, endothelial cells and smooth muscle cells of arteries and veins, endothelial cells of liver sinusoids and certain capillaries, uterine smooth muscle cells, myoepithelial cells, perineurial cells of peripheral nerves and “fibroblastic” cells of granulation tissue. The specificity of immunohistologic staining was confirmed by appropriate absorption and blocking studies and immunodiffusional analysis in agarose gel. It was also observed by immunodiffusional analysis in agarose gel, electrophoresis of actomyosin fragments in polyacrylamide gels, immune inhibition of actomyosin ATPase activity and immune aggregation of platelets that uterine and platelet actomyosin are partially, but not completely, identical.     

含姜黄素的(丙交酯-乙交酯)共聚物薄膜对血管平滑肌细胞增殖的影响

血管支架内再狭窄一直是困扰经皮冠状动脉介入治疗的主要问题,药物洗脱支架的临床应用显著降低了术后再狭窄率而成为冠心病介入治疗新的里程碑.由于姜黄素具有抗炎和抗增生等药理活性而有望应用于药物洗脱支架.本文采用四唑蓝比色法测定了大鼠血管平滑肌细胞对不同浓度姜黄素的药物敏感性,实验结果表明,姜黄素的浓度范围为2.5～10 μg/ml时,对平滑肌细胞的抑制率大于80%.再以可降解高分子材料丙交酯-乙交酯共聚物(poly (lactic acid-co-glycol acid),PLGA)为载体分别制备了三种姜黄素浓度(3wt%,5wt%,8wt%)的复合薄膜.乳酸脱氢酶的释放检测结果显示这三种载药薄膜对平滑肌细胞没有急性的毒性反应.采用Alamar Blue法分别研究了三种浓度含姜黄素的PLGA薄膜和平滑肌细胞经3 d和7 d共培养后的细胞活性,表明三种浓度的含姜黄素薄膜均能够显著抑制平滑肌细胞增殖,尤其当薄膜中姜黄素浓度为8wt%时,细胞活性值最低,抑制作用最明显.     

Chlamydia pneumoniae and Chlamydia Trachomatis Infection Differentially Modulates Human Dendritic Cell Line (MUTZ) Differentiation and Activation

Chlamydia trachomatis and Chlamydia pneumoniae are important human pathogens that infect the urogenital/anorectal and respiratory tracts, respectively. Whilst the ability of these bacteria to infect epithelia is well defined, there is also considerable evidence of infection of leucocytes, including dendritic cells (DCs). Using a human dendritic cell line (MUTZ), we demonstrate that the infection and replication of chlamydiae inside DCs is species and serovar specific and that live infection with C. pneumoniae is required to upregulate costimulatory markers CD80, CD83 and human leucocyte antigen (HLA)‐DR on MUTZ cells, as well as induce secretion of interleukin (IL)‐2, IL‐6, IL‐8, IL‐12 (p70), interferon‐gamma and tumour necrosis factor‐alpha Conversely, C. trachomatis serovar D failed to upregulate DC costimulatory markers, but did induce secretion of high concentrations of IL‐8. Interestingly, we also observed that infection of MUTZ cells with C. pneumoniae or C. trachomatis serovar L2, whilst not replicative, remained infectious and upregulated lymph node migratory marker CCR7 mRNA. Taken together, these data confirm the findings of other groups using primary DCs and demonstrate the utility of MUTZ cells for further studies of chlamydial infection.     

细胞外信号调节激酶在缓激肽引起血管平滑肌细胞增殖反应中的调节作用

目的 :本研究主要从细胞外信号调节激酶 (ERKs)的角度 ,研究丝裂原活化蛋白激酶 (MAPK)信号途径在缓激肽 (BK)介导的大鼠血管平滑肌细胞 (VSMC)促增殖等反应中的作用及其可能的调控机制。方法 :通过3H 胸苷 ( 3H TdR)掺入率与SMC3H 亮氨酸掺入率分别反映SMC的DNA代谢与蛋白质合成代谢速率 ;并通过给予MAPK激酶 (MEK )特异性抑制剂PD0 980 5 9及ERKS抑制剂UO12 6预处理 ,观察它们对细胞蛋白合成和DNA代谢及细胞增殖的影响。结果 :①BK( 10 - 8mmol/L)处理 3 0minVSMC3H 胸苷掺入率和3H 亮氨酸掺入率均明显增高 ;②BK增高3H 胸苷掺入的作用可明显被PD0 980 5 9所抑制和部分被UO12 6所抑制 ;③BK增高3H 亮氨酸掺入的作用可部分被PD0 980 5 9和UO12 6所抑制。结论 :ERKs激活在缓激肽导致VSMC增殖反应中具有重要作用 ,并可通过ERKs信号途径的特异性抑制剂的抑制效应 ,影响血管平滑肌细胞的增殖效应     

串珠素对大鼠平滑肌细胞增殖的影响及其机制的研究

目的 观察串珠素对于大鼠血管平滑肌细胞（vascular smooth muscle cells。VSMCs）增殖反应的影响以及细胞信号转导机制。方法以^3H-TdR掺人法测定VSMCs增殖。Westem blot方法检测FAK表达。结果 Perlecan加强碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF）引起的VSMCs增殖反应，并诱导粘着斑激酶（focal adhesion kinase，FAK）表达上调。Perlecan抗体消除Perlecan促进bFGF致VSMCs增殖反应，并抑制FAK表达。结论 串珠素增强bFGF致大鼠平滑肌细胞的增殖反应，其机制涉及FAK表达上调。     

可溶性血管内皮生长因子受体1真核克隆表达及其对血管内皮细胞增殖的影响

本研究旨在构建可溶性血管内皮生长因子(vascular endothelial growth factor,VEGF)受体1(soluble fmslike tyosine kinase-1,sFlt-1)的真核表达质粒pcDNA3.1-sFlt-1,并观察sFlt-1对血管内皮细胞增殖的影响。提取人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)总RNA,扩增Flt-1基因胞外1-3结构域,构建真核表达质粒pcDNA3.1-sFlt-1,测序鉴定基因序列。将重组质粒转染Lewis肺癌细胞,采用RT-PCR和SDS-PAGE检测sFlt-1在基因及蛋白水平的表达情况。MTT法检测sFlt-1对VEGF诱导的HUVECs生长的影响。结果显示:①插入片段序列正确;②sFlt-1在基因水平成功表达且转染后的Lewis肺癌细胞能分泌表达sFlt-1;③含sFlt-1的细胞上清液可明显抑制VEGF诱导的HUVECs增殖。本研究成功构建了真核表达质粒pcDNA3.1-sFlt-1,sFlt-1,在基因和蛋白水平均获得有效表达,且表达的蛋白可明显抑制由VEGF诱导...     

血小板源生长因子在低氧性肺动脉平滑肌细胞增殖中的作用

运用流式细胞仪及细胞原位杂交方法测定低氧对培养的新生牛肺动脉平滑肌细胞（ＰＡＳＭＣ）生长的影响，并探讨血小板源生长因子（ＰＤＧＦ）在其中的作用，结果表明：培养的ＰＡＳＭＣ的低氧１２ｈ时，由Ｇ０／Ｇ１期进入Ｓ斯的细胞数明显增多，为对照组的１５７．７％（Ｐ〈０．０５）；ＰＤＧＦ可进一步促进低氧状态下ＰＡＳＭＣ的增殖，Ｓ期细胞为对照组的２４２．３％（Ｐ〈０．０１），原位杂交结果显示：低氧（１２ｈ）条件下     

生长抑素抑制内皮素刺激的家兔血管平滑肌细胞增殖

为探讨生长抑素（ＳＳＴ）对内皮素（ＥＴ）刺激细胞增殖的作用其机理，本工作在培养的兔主动脉血管平滑肌细胞（ＶＳＭＣ）上发现，１０＾－８ｍｏｌ／ＬＥＴ刺激细胞增殖（＾３Ｈ＝ＴｄＲ参入增多）和丝裂素活化蛋白激酶（ＭＡＰＫ）激活，１０＾－８ｍｏｌ／ＬＳＳＴ单独作用可抑制细胞增玩具增殖但不曩ＭＡＰＫ活性。ＳＳＴ不仅呈浓度依赖性地抑制ＥＴ刺激的ＶＳＭＣ增殖（Ｐ〈０．０１），而且亦抑制ＥＴ刺激的细胞ＭＡＰＫ激     

降钙素基因相关肽抑制大鼠主动脉平滑肌细胞增殖

本工作用培养的大鼠主动脉平滑肌细胞，以细胞计数和＾３Ｈ－胸腺嘧啶掺入为指标，观察到降钙素基因相关肽呈剂量依赖地抑制ＶＳＭＣ增殖，１０＾－８和１０＾－７ｍｏｌ／ＬＣＧＲＰ可使＾３Ｈ－ＴｄＲ掺入量较对照组减少２６％（Ｐ＜０．０５）和３６％（Ｐ＜０．０１）并使细胞计数分别较对照组下降１７％（Ｐ＜０．０５）和３５％（Ｐ＜０．０１）。提示ＣＧＲＰ参与ＶＳＭＣ增殖的调节。     

Epithelial Cells Infected with Chlamydophila pneumoniae (Chlamydia pneumoniae) Are Resistant to Apoptosis

The obligate intracellular pathogen Chlamydophila pneumoniae (Chlamydia pneumoniae) initiates infections in humans via the mucosal epithelia of the respiratory tract. Here, we report that epithelial cells infected with C. pneumoniae are resistant to apoptosis induced by treatment with drugs or by death receptor ligation. The induction of protection from apoptosis depended on the infection conditions since only cells containing large inclusions were protected. The underlying mechanism of infection-induced apoptosis resistance probably involves mitochondria, the major integrators of apoptotic signaling. In the infected cells, mitochondria did not respond to apoptotic stimuli by the release of apoptogenic factors required for the activation of caspases. Consequently, active caspase-3 was absent in infected cells. Our data suggest a direct modulation of apoptotic pathways in epithelial cells by C. pneumoniae.     

Phagocytosis of Apoptotic Trophoblast Cells by Human Endometrial Endothelial Cells Induces Proinflammatory Cytokine Production

Citation Peng B, Koga K, Cardenas I, Aldo P, Mor G. Phagocytosis of apoptotic trophoblast cells by human endometrial endothelial cells induces proinflammatory cytokine production. Am J Reprod Immunol 2010; 64: 12–19 Problem Apoptosis is a normal constituent of trophoblast turnover in the placenta; however in some cases, this process is related to pregnancy complications such as preeclampsia. Recognition and engulfment of these apoptotic trophoblast cells is important for clearance of dying cells. The aim of this study was to show the cross talk between human endometrial endothelial cells (HEECs) and apoptotic trophoblast cells in an in vitro coculture model and its effect on cytokine production by HEECs. Method of study Fluorescent‐labeled HEECs were cocultured with fluorescent‐labeled apoptotic human trophoblast cells. Confocal microscopy and flowcytometry were used to show the interaction between these two types of cells. Cytokine profiles were determined using multiplex analysis. Results HEECs are capable to phagocytose apoptotic trophoblasts. This activity is inhibited by the phagocytosis inhibitor cytochalasin B. Phagocytosis of apoptotic trophoblast cells induced the secretion of the proinflammatory cytokines interleukin‐6 and monocyte chemoattractant protein‐1 by HEECs. Conclusion This study provides the first evidence that HEECs have an ability to phagocytose apoptotic trophoblasts. Furthermore, we demonstrated an inflammatory response of HEECs after phagocytosing the apoptotic trophoblast cells. This event may contribute to the inflammatory response in both normal pregnancy and pathologic pregnancy such as preeclampsia.     

重组ACE2抑制人脐动脉平滑肌细胞Profilin-1表达及增殖

 摘要 目的：探讨重组血管紧张素转换酶2（ACE2）基因对血管平滑肌细胞前纤维蛋白-1（Profilin-1）表达及细胞增殖的影响。方法：培养人脐动脉平滑肌细胞（HUASMC），用重组ACE2基因（rACE2）干预，进行血管紧张素II（Ang II）刺激实验，分别用MTT法、实时定量PCR及Western blot检测HUASMC增殖与Profilin-1表达。 结果：与对照组相比，经Ang II（100 nmol /L）刺激6 h后，HUASMC中Profilin-1 表达明显增高（3.50±0.30 vs. 1.00±0.10, P<0.05）。而重组ACE2基因干预显著抑制上述增加（1.73±0.12 vs. 3.50±0.30, P<0.05）。结论：ACE2基因过表达可明显逆转HUASMC中Ang II诱导的Profilin-1表达上调。     

淋巴细胞对人主动脉平滑肌细胞迁移的影响

用微孔滤膜法分别检测淋巴细胞条件培养液（ＬＣＭ）和γ－ＩＦＮ对培养的人主动脉平滑肌细胞（ＳＭＣ）的趋化活性。结果表明，ＬＣＭ对ＳＭＣ有明显的趋化活性和较弱的化学促动作用；γ－ＩＦＮ对ＳＭＣ有明显的趋化作用和化学促动作用，在５００Ｕ／ｍｌ浓度时其活性最强。提示动脉粥样硬化早期病灶中激活的Ｔ淋巴细胞可通过分泌γ－ＩＦＮ（或伴有其它因子），吸引中膜的ＳＭＣ迁入内膜。