SDF-1-CXCR4轴与缺血性心脏病的干细胞治疗

干细胞治疗为缺血性心脏病患者提供了一种简单、经济、有效的全新治疗方法,其早期临床实验也取得了令人振奋的结果,然而更广泛的临床应用亟需更深入的基础研究的支持。SDF-1-CXCR4轴在干细胞的动员和归巢中起着关键作用,将SDF-1与干细胞治疗结合将为缺血性心脏病的细胞治疗提供一个新的途径。本文就缺血性心脏病的干细胞治疗,SDF-1-CXCR4轴在干细胞动员和心脏损伤、修复中的作用作一综述。     

Cdk4 deficiency inhibits skin tumor development but does not affect normal keratinocyte proliferation

Most human tumors have mutations that result in deregulation of the cdk4/cyclin-Ink4-Rb pathway. Overexpression of D-type cyclins or cdk4 and inactivation of Ink4 inhibitors are common in human tumors. Conversely, lack of cyclin D1 expression results in significant reduction in mouse skin and mammary tumor development. However, complete elimination of tumor development was not observed in these models, suggesting that other cyclin/cdk complexes play an important role in tumorigenesis. Here we described the effects of cdk4 deficiency on mouse skin proliferation and tumor development. Cdk4 deficiency resulted in a 98% reduction in the number of tumors generated through the two-stage carcinogenesis model. The absence of cdk4 did not affect normal keratinocyte proliferation and both wild-type and cdk4 knockout epidermis are equally affected after topical treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), resulting in epidermal hyperplasia. In similar fashion, cdk4 knockout keratinocytes proliferated well in an in vivo model of wound-induced proliferation. Biochemical studies in mouse epidermis showed that cdk6 activity increased twofold in cdk4-deficient mice compared to wild-type siblings. These results suggest that therapeutic approaches to inhibit cdk4 activity could provide a target to inhibit tumor development with minimal or no effect in normal tissue.     

Trafficking of normal stem cells and metastasis of cancer stem cells involve similar mechanisms: pivotal role of the SDF-1-CXCR4 axis

The alpha-chemokine stromal-derived factor (SDF)-1 and the G-protein-coupled seven-span transmembrane receptor CXCR4 axis regulates the trafficking of various cell types. In this review, we present the concept that the SDF-1-CXCR4 axis is a master regulator of trafficking of both normal and cancer stem cells. Supporting this is growing evidence that SDF-1 plays a pivotal role in the regulation of trafficking of normal hematopoietic stem cells (HSCs) and their homing/retention in bone marrow. Moreover, functional CXCR4 is also expressed on nonhematopoietic tissue-committed stem/progenitor cells (TCSCs); hence, the SDF-1-CXCR4 axis emerges as a pivotal regulator of trafficking of various types of stem cells in the body. Furthermore, because most if not all malignancies originate in the stem/progenitor cell compartment, cancer stem cells also express CXCR4 on their surface and, as a result, the SDF-1-CXCR4 axis is also involved in directing their trafficking/metastasis to organs that highly express SDF-1 (e.g., lymph nodes, lungs, liver, and bones). Hence, we postulate that the metastasis of cancer stem cells and trafficking of normal stem cells involve similar mechanisms, and we discuss here the common molecular mechanisms involved in these processes. Finally, the responsiveness of CXCR4+ normal and malignant stem cells to an SDF-1 gradient may be regulated positively/primed by several small molecules related to inflammation which enhance incorporation of CXCR4 into membrane lipid rafts, or may be inhibited/blocked by small CXCR4 antagonist peptides. Consequently, strategies aimed at modulating the SDF-1-CXCR4 axis could have important clinical applications both in regenerative medicine to deliver normal stem cells to the tissues/organs and in clinical hematology/oncology to inhibit metastasis of cancer stem cells.     

Cryopreservation does not affect proliferation and multipotency of murine neural precursor cells

Stem cell research offers unique opportunities for developing new medical therapies for devastating diseases and a new way to explore fundamental questions of biology. Establishing an efficient freezing protocol for neural precursor cells (NPCs) is of great importance for advances in cell-based therapies. We used fluorescence-activated cell sorter-based cell death/survival analysis and Western blot analysis of proliferation markers (proliferating cell nuclear antigen) and prosurvival proteins (Bcl-2) to study the effect of a variety of cryoprotective agents on fetal mouse forebrain NPCs. Neurospheres frozen at -70 degrees C or in liquid nitrogen in a rate-controlled manner and thawed after 5 days retained viability of 60%-70% measured 24 hours after thawing. However, 1 week after thawing, viability dropped to 50%-60%. Using a clonogenic sphere formation assay, we showed that recovery rate of frozen NPCs was approximately 26% and did not significantly differ between dimethyl sulfoxide (DMSO)- and glycerol-supplemented samples. Application of the caspase inhibitor zVAD-fmk during freezing or in the first week after thawing resulted in protection of cryopreserved neurospheres after thawing but not during the freezing process, indicating that apoptosis limits recovery of NPCs. Cell survival was not reduced in cells that were enzymatically separated before cryopreservation. Optimal protection of NPCs was achieved when 10% DMSO alone or in a combination with 10% fetal calf serum (FCS) was used. However, 10% glycerol alone was equally effective. Using these protocols, NPCs retained their multipotency and differentiated into both glial (GFAP-positive) and neuronal (Tuj1-positive) cells. Percentage of Tuj1-positive cells in 5% and 10% DMSO, in 10% DMSO + 10% FCS, and in 10% glycerol remained at the same level as before freezing and varied from 5%-7%. We conclude that cryopreservation (up to 1 month at -70 degrees C and up to 1 year in liquid nitrogen) does not markedly alter the rate of proliferation and multipotency of murine neural precursor cells.     

CDK2 activation in mouse epidermis induces keratinocyte proliferation but does not affect skin tumor development

It has been widely assumed that elevated CDK2 kinase activity plays a contributory role in tumorigenesis. We have previously shown that mice overexpressing CDK4 under control of the keratin 5 promoter (K5CDK4 mice) develop epidermal hyperplasia and increased susceptibility to squamous cell carcinomas. In this model, CDK4 overexpression results in increased CDK2 activity associated with the noncatalytic function of CDK4, sequestration of p21(Cip1) and p27(Kip1). Furthermore, we have shown that ablation of Cdk2 reduces Ras-Cdk4 tumorigenesis, suggesting that increased CDK2 activity plays an important role in Ras-mediated tumorigenesis. To investigate this hypothesis, we generated two transgenic mouse models of elevated CDK2 kinase activity, K5Cdk2 and K5Cdk4(D158N) mice. The D158N mutation blocks CDK4 kinase activity without interfering with its binding capability. CDK2 activation via overexpression of CDK4(D158N), but not of CDK2, resulted in epidermal hyperplasia. We observed elevated levels of p21(Cip1) in K5Cdk2, but not in K5Cdk4(D158N), epidermis, suggesting that CDK2 overexpression elicits a p21(Cip1) response to maintain keratinocyte homeostasis. Surprisingly, we found that neither CDK2 overexpression nor the indirect activation of CDK2 enhanced skin tumor development. Thus, although the indirect activation of CDK2 is sufficient to induce keratinocyte hyperproliferation, activation of CDK2 alone does not induce malignant progression in Ras-mediated tumorigenesis.     

SDF-1/CXCR4轴活化诱导内皮祖细胞增殖、迁移及管型形成

目的观察趋化因子SDF-1促内皮祖细胞增殖、迁移和管型形成的作用。方法用免疫细胞化学检测内皮祖细胞SDF-1和CXCR4表达;用MTT法、Millicell趋化法及Matrigel体外三维成型法分别检测不同浓度的趋化因子SDF-1促内皮祖细胞增殖、迁移和管型形成。并应用CXCR4受体抑制剂AMD3100观察上述指标的变化。结果免疫细胞化学显示内皮祖细胞表达SDF-1和CXCR4蛋白。SDF-1可促进内皮祖细胞的增殖、迁移和体外小管样结构的形成。AMD3100可抑制SDF-1的诱导作用。结论SDF-1/CXCR4轴在内皮祖细胞参与血管新生中可能发挥重要作用。     

Interferon-gamma stimulates the secretion of IL-1, but not of IL-6, by glomerular mesangial cells.

IL-1 activity in culture supernatant and cell lysate from rat mesangial cells stimulated with interferon-gamma (IFN-gamma) was measured by a thymocyte proliferation assay. While IFN-gamma alone had no effect on the secretion or the intracellular pool of IL-1, the enhancement by IFN-gamma of IL-1 secretion in response to lipopolysaccharide (LPS) was observed. The stimulatory effect of culture supernatant on thymocyte proliferation was abrogated by preincubation with the anti-IL-1 antibody. At least 4-h incubation with IFN-gamma and LPS was required to detect enhancing effect of IFN-gamma. The addition of as little as 1 U/ml IFN-gamma significantly increased IL-1 secretion in the presence of 10 micrograms/ml LPS. The IL-6 activity in culture supernatants was determined by measurement of thymidine uptake in mouse IL-6-dependent cell line (MH60.BSF2). Mesangial cells secreted IL-6 in culture supernatant without additional stimuli and LPS distinctly increased it as described previously. However, in contrast to IL-1 production, no effect of IFN-gamma on IL-6 secretion was observed in the presence or absence of LPS. Moreover, we determined whether enhanced IL-1 release is associated with Ia expression on mesangial cells. IFN-gamma alone and the combination with LPS induced marked expression of Ia antigen, whereas LPS alone did not. We conclude that IFN-gamma stimulates the production of IL-1, but not IL-6, by mesangial cells and suggest an important role of IFN-gamma in the pathogenesis of glomerulonephritis by regulating the mesangial production of IL-1 and the accessory cell function of mesangial cells.     

Wnt1 and Wnt5a affect endothelial proliferation and capillary length; Wnt2 does not

Blood vessel growth is critical for embryonic development and contributes to pathologies including cancer and diabetic retinopathy. A growing body of evidence suggests that signaling via the Wnt/β-catenin pathway contributes to angiogenesis, and that paracrine Wnt signaling might alter endothelial cell function. To test the hypothesis that Wnt signaling promotes endothelial cell proliferation and vessel growth, we treated bovine aortic endothelial cells with Wnt1, Wnt2 and Wnt5a derived from coculture with Wnt-expressing fibroblasts. Endothelial cells cultured in the presence of Wnt1 displayed increased Wnt/β-catenin signaling, proliferation and capillary stability in vitro. Wnt5a, which primarily signals via an alternate Wnt pathway, the Wnt/Ca⁺⁺ pathway, decreased both cell number and capillary length. Wnt2, which in other cell types activates the Wnt/β-catenin pathway, did not activate signaling, affect cell number or increase capillary length. These results suggest that Wnt/β-catenin and Wnt/Ca⁺⁺ signals might have opposing effects on angiogenesis.     

Wnt1 and Wnt5a affect endothelial proliferation and capillary length; Wnt2 does not

Blood vessel growth is critical for embryonic development and contributes to pathologies including cancer and diabetic retinopathy. A growing body of evidence suggests that signaling via the Wnt/beta-catenin pathway contributes to angiogenesis, and that paracrine Wnt signaling might alter endothelial cell function. To test the hypothesis that Wnt signaling promotes endothelial cell proliferation and vessel growth, we treated bovine aortic endothelial cells with Wnt1, Wnt2 and Wnt5a derived from coculture with Wnt-expressing fibroblasts. Endothelial cells cultured in the presence of Wnt1 displayed increased Wnt/beta-catenin signaling, proliferation and capillary stability in vitro. Wnt5a, which primarily signals via an alternate Wnt pathway, the Wnt/Ca(++) pathway, decreased both cell number and capillary length. Wnt2, which in other cell types activates the Wnt/beta-catenin pathway, did not activate signaling, affect cell number or increase capillary length. These results suggest that Wnt/beta-catenin and Wnt/Ca(++) signals might have opposing effects on angiogenesis.     

Gliomatosis peritonei is associated with frequent recurrence, but does not affect overall survival in patients with ovarian immature teratoma

Gliomatosis peritonei (GP) is commonly associated with ovarian teratoma and is not thought to have an adverse prognostic effect. However, the prognostic impact and characteristics of GP remain to be clarified. In this study, we investigated the clinicopathologic features of ovarian teratoma associated with GP, and we further compared ovarian immature teratoma (IT) with GP to ovarian IT without GP. During the study period, there were a total of 16 ovarian teratomas associated with GP. Among them, 15 cases were ovarian ITs of various grades. When ovarian IT with GP (n?=?15) was compared to ovarian IT without GP (n?=?27), it was found that ovarian IT patients with GP had larger tumor size (median, 19 vs. 13?cm; P?相似文献   

XLP1 inhibitory effect by 2B4 does not affect DNAM‐1 and NKG2D activating pathways in NK cells

X‐linked lymphoproliferative disease 1 (XLP1) is a rare congenital immunodeficiency caused by SH2D1A (Xq25) mutations resulting in lack or dysfunction of SLAM‐associated protein adaptor molecule. In XLP1 patients, upon ligand (CD48) engagement, 2B4 delivers inhibitory signals that impair the cytolytic activity of NK (and T) cells. This causes the selective inability to control EBV infections and the occurrence of B‐cell lymphomas. Here, we show that in the absence of SLAM‐associated protein, co‐engagement of 2B4 with different activating receptors, either by antibodies or specific ligands on target cells, inhibits different ITAM‐dependent signaling pathways including activating killer Ig‐like receptors. In XLP1 NK cells, 2B4 affected both the cytolytic and IFN‐γ production capabilities, functions that were restored upon disruption of the 2B4/CD48 interactions. Notably, we provide evidence that 2B4 dysfunction does not affect the activity of DNAM‐1 and NKG2D triggering receptors. Thus, while CD48⁺ B‐EBV and lymphoma B cells devoid of NKG2D and DNAM‐1 ligands were resistant to lysis, the preferential usage of these receptors allowed XLP1 NK cells to kill lymphomas that expressed sufficient amounts of the specific ligands. The study sheds new light on the XLP1 immunological defect and on the cross‐talk of inhibitory 2B4 with triggering NK (and T) receptors.     

Myosin VI in PC12 cells plays important roles in cell migration and proliferation but not in catecholamine secretion

Myosin VI (MVI) is the only known myosin walking towards minus end of actin filaments and is believed to play distinct role(s) than other myosins. We addressed a role of this unique motor in secretory PC12 cells, derived from rat adrenal medulla pheochromocytoma using cell lines with reduced MVI synthesis (produced by means of siRNA). Decrease of MVI expression caused severe changes in cell size and morphology, and profound defects in actin cytoskeleton organization and Golgi structure. Also, significant inhibition of cell migration as well as cell proliferation was observed. Flow cytometric analysis revealed that MVI-deficient cells were arrested in G0/G1 phase of the cell cycle but did not undergo increased senescence as compared with control cells. Also, neither polyploidy nor aneuploidy were detected. Surprisingly, no significant effect on noradrenaline secretion was observed. These data indicate that in PC12 cells MVI is involved in cell migration and proliferation but is not crucial for stimulation-dependent catecholamine release.     

Stromal-derived factor-1alpha/CXCL12-CXCR 4 axis is involved in the dissemination of NSCLC cells into pleural space

Malignant pleural effusion (PE) is one of the poor prognostic factors in non-small cell lung cancer (NSCLC), and the detailed mechanism of the malignant PE formation is not fully elucidated. Recently, CXCR4, a receptor for chemokine stromal-derived factor-1alpha (SDF-1alpha) that can induce chemotaxis of cells, was reported to be expressed on NSCLC. In this study, we hypothesized that the SDF-1alpha/CXCR4 axis may be involved in the dissemination of malignant cells into pleural space, and investigated its expression, function, and signaling pathway using NSCLC cell lines and clinical samples from 43 patients with NSCLC with malignant PE. We found functional expression of CXCR4 on NSCLC cell lines, and also found that SDF-1alpha could induce migration via phosphatidylinositol 3 (PI-3) kinase- and p44/42 mitogen-activated protein kinase-dependent manner. The SDF-1alpha levels in malignant PE were significantly higher than those in transudate PE and showed a significant positive correlation with PE volumes. The sensitivity and specificity for prediction of recurrence of malignant PE was 61.5% and 83.3%, respectively (cutoff SDF-1alpha = 2,500 ng/ml), and better than those using pH of PE. Cancer cells in malignant PE expressed CXCR4, and mesothelial cells of the pleura stained positive for SDF-1alpha. The SDF-1alpha/CXCR4 axis is involved in the dissemination of NSCLC cells into pleural space.     

Type I Helicobacter pylori lipopolysaccharide stimulates toll-like receptor 4 and activates mitogen oxidase 1 in gastric pit cells

Guinea pig gastric pit cells express an isozyme of gp91-phox, mitogen oxidase 1 (Mox1), and essential components for the phagocyte NADPH oxidase (p67-, p47-, p40-, and p22-phox). Helicobacter pylori lipopolysaccharide (LPS) and Escherichia coli LPS have been shown to function as potent activators for the Mox1 oxidase. These cells spontaneously secreted about 10 nmol of superoxide anion (O(2)(-))/mg of protein/h under LPS-free conditions. They expressed the mRNA and protein of Toll-like receptor 4 (TLR4) but not those of TLR2. LPS from type I H. pylori at 2.1 endotoxin units/ml or higher stimulated TLR4-mediated phosphorylations of transforming growth factor beta-activated kinase 1 and its binding protein 1 induced TLR4 and p67-phox and up-regulated O(2)(-) production 10-fold. In contrast, none of these events occurred with H. pylori LPS from complete or partial deletion mutants of the cag pathogenicity island. Lipid A was confirmed to be a bioactive component for the priming effects, while removal of bisphosphates from lipid A completely eliminated the effects, suggesting the importance of the phosphorylation pattern besides the acylation pattern for the bioactivity. H. pylori LPS is generally accepted as having low toxicity; however, our results suggest that type I H. pylori lipid A may be a potent stimulator for innate immune responses of gastric mucosa by stimulating the TLR4 cascade and Mox1 oxidase in pit cells.     

Osteopontin stimulates invasion of NCI‐h295 cells but is not associated with survival in adrenocortical carcinoma

Gene array studies indicated that osteopontin (OPN) mRNA is highly expressed in adrenocortical carcinomas (ACCs). OPN enhances invasiveness, proliferation, and metastasis formation, and is associated with poor survival in some malignant diseases. Integrin αvβ3 has been shown to mediate OPN effects on invasion. In this study, we demonstrated OPN and integrin αvβ3 expression in normal adrenal glands and benign adenomas, with staining seen exclusively in adrenocortical cells as well as even stronger staining in ACC. Western blot analysis confirmed overexpression of OPN in ACC (p < 0.01). With Matrigel invasion assays, we have shown that OPN greatly stimulates the invasiveness of NCI‐h295 cells (>six‐fold increase, p < 0.001). Transfection with integrin αvβ3 further increased invasiveness after OPN stimulation (p < 0.001). This increase was reversed by the addition of an anti‐integrin β3 antibody, indicating a functional relationship of OPN and integrin αvβ3 in ACC. With tissue arrays, we confirmed high OPN expression in 147 ACC samples. However, no association with survival was seen in Kaplan‐Meier analysis including 111 patients with primary tumours graded for OPN staining and follow‐up data available. In conclusion, our in vitro data indicate that OPN and integrin αvβ3 may act as a functional complex facilitating the invasiveness of adrenocortical tumours. This relationship remains of relevance to our understanding of carcinogenesis, but further studies are needed to address the physiological and pathological function of OPN in adrenal tissue. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.