Role of PCK2 in the proliferation of vascular smooth muscle cells in neointimal hyperplasia

Vascular smooth muscle cell (VSMC) proliferation is a hallmark of neointimal hyperplasia (NIH) in atherosclerosis and restenosis post-balloon angioplasty and stent insertion. Although numerous cytotoxic and cytostatic therapeutics have been developed to reduce NIH, it is improbable that a multifactorial disease can be successfully treated by focusing on a preconceived hypothesis. We, therefore, aimed to identify key molecules involved in NIH via a hypothesis-free approach. We analyzed four datasets ({"type":"entrez-geo","attrs":{"text":"GSE28829","term_id":"28829"}}GSE28829, {"type":"entrez-geo","attrs":{"text":"GSE43292","term_id":"43292"}}GSE43292, {"type":"entrez-geo","attrs":{"text":"GSE100927","term_id":"100927"}}GSE100927, and {"type":"entrez-geo","attrs":{"text":"GSE120521","term_id":"120521"}}GSE120521), evaluated differentially expressed genes (DEGs) in wire-injured femoral arteries of mice, and determined their association with VSMC proliferation in vitro. Moreover, we performed RNA sequencing on platelet-derived growth factor (PDGF)-stimulated human VSMCs (hVSMCs) post-phosphoenolpyruvate carboxykinase 2 (PCK2) knockdown and investigated pathways associated with PCK2. Finally, we assessed NIH formation in Pck2 knockout (KO) mice by wire injury and identified PCK2 expression in human femoral artery atheroma. Among six DEGs, only PCK2 and RGS1 showed identical expression patterns between wire-injured femoral arteries of mice and gene expression datasets. PDGF-induced VSMC proliferation was attenuated when hVSMCs were transfected with PCK2 siRNA. RNA sequencing of PCK2 siRNA-treated hVSMCs revealed the involvement of the Akt-FoxO-PCK2 pathway in VSMC proliferation via Akt2, Akt3, FoxO1, and FoxO3. Additionally, NIH was attenuated in the wire-injured femoral artery of Pck2-KO mice and PCK2 was expressed in human femoral atheroma. PCK2 regulates VSMC proliferation in response to vascular injury via the Akt-FoxO-PCK2 pathway. Targeting PCK2, a downstream signaling mediator of VSMC proliferation, may be a novel therapeutic approach to modulate VSMC proliferation in atherosclerosis.     

APP5肽模拟物P165对体外培养大鼠海马神经干细胞增殖和分化的影响

目的研究一种小分子多肽─APP5肽的模拟物P165对体外培养的大鼠胚胎海马神经干细胞（neuralstem cells,NSCs）增殖和分化的影响,以期能找到一种可代替神经营养因子的小分子物质,能够促进NSCs的增殖或分化,为将来的临床应用提供理论依据。方法（1）原代培养SD大鼠胚胎脑海马NSCs;（2）利用5-溴脱氧尿嘧啶核苷（BrdU）和神经元、星型胶质细胞、少突胶质细胞的特异性标记物微管相关蛋白2（MAP2）、胶质纤维酸性蛋白（GFAP）、2,3-环核苷酸-3磷酸二酯酶（CNPase）对培养的NSCs进行鉴定;（3）将培养的NSCs分为对照组、血清组、APP5肽反序列组和P165组,观察各组细胞形态的变化;（4）将培养的NSCs分为对照组、APP5肽反序列组和P165组,利用细胞计数,测定干细胞克隆形成率、干细胞克隆形成大小的方法分析P165对海马NSCs增殖的影响。结果（1）海马神经干细胞呈神经球聚集生长,BrdU染色阳性;加入血清后神经球周围有细胞呈放射状向四周生长,并带有突起。染色呈MAP2、GFAP或CNPase阳性;（2）海马NSCs加入P165及其反序列后细胞形态上与对照组相比没有明显改变;（3）与对照组相比,加P165后海马NSCs数量明显增加,克隆形成率和克隆形成的直径均有明显的增加,并有统计学差异。结论P165能够促进海马NSCs的增殖,但并不促进其分化。     

低氧对胚胎干细胞增殖的影响

目的:观察间歇性低氧和持续性低氧对体外培养的胚胎干细胞(ES细胞)增殖的影响.方法:利用细胞记数法和BrdU (5-溴脱氧尿苷)掺入的流式细胞分析检测细胞增殖,并用RT -PCR的方法检测低氧诱导因子(HIF-1a)的表达变化.结果:①将ES细胞分别放在低氧(3%～10% O2)和常氧(20% O2)的环境中培养24 h后,在低氧环境中培养的ES细胞数较常氧组明显减少;②将ES细胞分别给予间歇性低氧刺激(3%～10% O2),每天10 min,连续4 d后,发现3%低氧组较常氧对照组的细胞增殖明显升高.③用RT-PCR方法观察HIF-1a的表达与细胞增殖的关系,发现在常氧环境中培养的ES即有HIF-1a的表达,ES细胞在持续低氧24 h或间歇性低氧(3%～10% O2)刺激4 d后对HIF-1a的表达均无明显影响.结论:间歇性低氧(3% O2)可明显促进体外培养的ES细胞增殖,而持续性低氧抑制ES细胞增殖,间歇性低氧(3% O2)刺激促进ES细胞增殖的机制尚有待于进一步的研究.     

胃缺血-再灌注对大鼠胃黏膜细胞凋亡和增殖的影响

本研究采用大鼠胃缺血-再灌注（gastricischemia-reperfusion,GI-R）模型（夹闭腹腔动脉30 min后再灌注）,通过组织学、免疫组化等方法,研究GI-R不同时间（0、0.5、1、3、6、24、48、72 h）对胃黏膜细胞凋亡和增殖的影响.结果发现,单纯缺血30 min胃黏膜损伤较轻,再灌注后损伤逐渐加重,胃黏膜的凋亡细胞迅速增加,而增殖细胞迅速减少;至再灌注后1 h达高峰;之后胃黏膜开始修复,凋亡细胞逐渐减少,增殖细胞逐渐增加;至再灌注后24 h胃黏膜细胞增殖达高峰;再灌注后72 h胃黏膜基本恢复正常.上述结果提示,在GI-R中,胃黏膜损伤主要由再灌注引起,凋亡细胞增加;然后胃黏膜启动自我修复机制,增殖细胞逐渐取代损伤细胞,3 d左右就可基本修复,表明胃黏膜细胞具有很强的自我修复能力.     

Knockdown of SLC34A2 inhibits cell proliferation,metastasis, and elevates chemosensitivity in glioma

Solute carrier 34 A2 (SLC34A2) is a member of SLC34 family that is a group of phosphate transporters. SLC34A2 has been reported to play critical roles in tumorigenesis and progression. However, the researches about the biological roles of SLC34A2 in glioma have not yet been reported. In this study, we analyzed the expression patterns of SLC34A2 in clinical glioma tumor tissues and cell lines. The results demonstrated that SLC34A2 was generally overexpressed in both glioma tissues and cell lines. To further investigate the roles of SLC34A2 in glioma, lentivirus containing specific SLC34A2 short hairpin RNA (sh-SLC34A2) was used to infect glioma cell lines U251 and U87 for the knockdown of SLC34A2. The following studies proved that SLC34A2 knockdown exhibited suppressive effects on cell proliferation and migration/invasion. SLC34A2 knockdown also inhibited epithelial-mesenchymal transition (EMT) phenotype, as evidenced by the increased E-cadherin expression, and the decreased N-cadherin and fibronectin expressions. Besides, knockdown of SLC34A2 enhanced the temozolomide (TMZ) sensitivity of U251 and U87 cells. In vivo tumorigenicity assay demonstrated that SLC34A2 knockdown inhibited tumor growth. Moreover, SLC34A2 knockdown suppressed the activation of epidermal growth factor receptor (EGFR)/PI3K/AKT signaling pathway in U87 cells. GW2974 (EGFR inhibitor) increased SLC34A2 knockdown-inhibited cell proliferation, migration/invasion, as well as enhanced SLC34A2 knockdown-increased the TMZ sensitivity of glioma cells. These findings suggested that SLC34A2 might be a new potential therapeutic target for the therapy of glioma patients.     

More insights into the CCN3/Connexin43 interaction complex and its role for signaling

Connexin43 (Cx43) forms gap junction channels but also serves as a signaling center by binding to proteins via its C‐terminus. We have previously demonstrated that transfection of Cx43 leads to significantly reduced proliferation of placental tumor cells through upregulating and binding of the growth regulator CCN3 (NOV) at the C‐terminus of Cx43. Here, we combined fluorescence resonance energy transfer (FRET), co‐immunoprecipitation and proliferation and expression assays to characterize the interaction complex of Cx43 and CCN3. FRET measurements confirmed the interaction of CCN3 with wild‐type Cx43 (amino acids 1‐382) and with mutants of Cx43 truncated at the C‐terminus resulting in Cx43 proteins of amino acids 1‐374, 1‐273, 1‐264, 1‐257 in 293T cells. These results matched the co‐immunoprecipitation data. Interestingly, although FRET revealed distinct efficiencies in interaction of Cx43 with CCN3 for all deletion constructs only wild‐type Cx43 and one deletion construct (1‐374) led to increased CCN3 expression. Only these interactions which were associated with increased CCN3 expression resulted in a reduced cell proliferation. Our study provides evidence that only defined binding properties between Cx43 and CCN3 leading to an upregulation of CCN3 are needed for signaling. Furthermore, the data obtained by FRET analysis allowed us to model the 3D structure of the C‐terminus of Cx43 interacting with CCN3. J. Cell. Biochem. 110: 129–140, 2010. © 2010 Wiley‐Liss, Inc.     

Genistein Inhibits the Activity of Kv1.3 Potassium Channels in Human T Lymphocytes

In the present study, the whole-cell patch-clamp technique was applied to follow the inhibitory effect of genistein — a tyrosine kinase inhibitor and a natural anticancer agent—on the activity of voltage-gated potassium channels Kv1.3 expressed in human T lymphocytes (TL). Obtained data provide evidence that genistein application in the concentration range of 1–80 μM reversibly decreased the whole-cell potassium currents in TL in a concentration-dependent manner to about 0.23 of the control value. The half-blocking concentration range of genistein was from 10 to 40 μM. The current inhibition was correlated in time with a significant decrease of the current activation rate. The steady-state activation of the currents was unchanged upon application of genistein, as was the inactivation rate. The inhibitory effect of genistein on the current amplitude and activation kinetics was voltage-independent. The current inhibition was not changed significantly in the presence of 1 mM of sodium orthovanadate, a tyrosine phosphatase inhibitor. Application of daidzein, an inactive genistein analogue, did not affect significantly either the current amplitudes or the activation kinetics. Possible mechanisms of the observed phenomena and their significance for genistein-induced inhibition of cancer cell proliferation are discussed.     

Description of epithelial granular cell in catshark spiral intestine: Immunohistochemistry and ultrastructure

We evaluated the histology of the spiral intestine of the blackmouth catshark (Galeus melastomus), a small shark distributed in the eastern Atlantic and Mediterranean Sea basin. Entire digestive tracts of 10 G. melastomus were studied using histochemical, immunohistochemical, and ultrastructural methods. Our studies identified a unique, large granular cell type in the intestinal epithelium. Transmission electron microscopy showed that the epithelial granular cell type made intimate contact, by means of junctional complexes, with adjacent epithelial and mucous cells. Several histochemical staining methods showed that the cytoplasmic granules were strongly eosinophilic. Immunostaining of intestinal sections revealed immunoreactivity of the granular cell to tumor necrosis factor-α (TNF-α) antibody. However, no reactivity to inducible-nitric oxide synthase (i-NOS), interleukin-6 (IL-6), interleukin IL-1β, lysozyme, serotonin 5-HT antibodies was detected.     

胶原样分子与肿瘤关系研究进展

细胞外基质不仅维持着体内细胞微环境的稳定,还在细胞的正常生长、增殖以及细胞之间的信号传导中起着重要作用。肿瘤发生时,基质中的分子组分发生了改变,这些改变朝着有利于肿瘤细胞生长侵袭的方向发展。在这个过程中,细胞外基质的主要成分在合成和分解上发生巨大变化,胶原分子便是其中之一,胶原分子作为细胞外基质中的主要成分,对细胞的黏附、运动、迁移等活动起着重要作用。随着研究的深入,发现越来越多的胶原分子参与了肿瘤的发生发展。基质中还存在着一些分子,它们在结构上和胶原蛋白一样含有三螺旋胶原结构域,在肿瘤的发生发展过程中同样发挥着重要作用。本文就包括胶原分子在内的含有胶原结构的分子在肿瘤中的作用做一综述。