Stem cells in tumor angiogenesis

Contribution from diverse tissue-specific stem cell types is required to create the cell populations necessary for the activation of angiogenesis and neovascular growth in cancer. Bone marrow (BM)-derived circulating endothelial progenitors (EPCs) that would differentiate to bona fide endothelial cells (ECs) were previously believed to be necessary for tumor angiogenesis. However, numerous recent studies demonstrate that EPCs are not needed for tumor angiogenesis and indicate EPCs to be artifactual rather than physiological. It is evident that tumor infiltrating hematopoietic cells produced by BM-residing hematopoietic stem cells (HSCs) may contribute to tumor angiogenesis in a paracrine manner by stimulating ECs or by remodeling the extracellular matrix. Therefore, identification of the various hematopoietic cell subpopulations that are critical for tumor angiogenesis and better understanding of their proangiogenic functions and mechanisms of action have potential therapeutic significance. Stem and progenitor cell subsets for also other vascular or perivascular cell types such as pericytes or mesenchymal/stromal cells may provide critical contributions to the growing neovasculature. Furthermore, we hypothesize that the existence of a yet undiscovered—and largely unsearched—tissue-specific adult vascular endothelial stem cell (VESC) would provide completely novel targeted approaches to block pathological angiogenesis and cancer growth. This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited".     

血管内皮细胞迁移与肿瘤血管生成

肿瘤生长和转移均依赖于肿瘤的血管生成,而血管内皮细胞迁移是肿瘤血管生成过程的一个重要环节,有着重要的研究意义.现从肿瘤血管生成过程中促血管生成因子、骨架蛋白解聚、细胞外基质的降解三个方面对血管内皮细胞迁移的影响进行综述.     

Nestin:A novel angiogenesis marker and possible target for tumor angiogenesis

Abnormal vasculature,termed tumor vessels,is a hallmark of solid tumors.The degree of angiogenesis is associated with tumor aggressiveness and clinical outcome.Therefore,exact quantification of tumor vessels is useful to evaluate prognosis.Furthermore,selective detection of newly formed tumor vessels within cancer tissues using specific markers raises the possibility of molecular targeted therapy via the inhibition of tumor angiogenesis.Nestin,an intermediate filament protein,is reportedly expressed in repair processes,various neoplasms,and proliferating vascular endothelial cells.Nestin expression is detected in endothelial cells of embryonic capillaries,capillaries of the corpus luteum,which replenishes itself by angiogenesis,and proliferating endothelial progenitor cells,but not in mature endothelial cells.Therefore,expression of nestin is relatively limited to proliferating vascular endothelial cells and endothelial progenitor cells.Nestin expression is also reported in blood vessels within glioblastoma,prostate cancer,colorectal cancer,and pancreatic cancer,and its expression is more specific for newly formed blood vessels than other endothelial cell markers.Nestin-positive blood vessels form smaller vessels with high proliferation activity in tumors.Knockdown of nestin in vascular endothelial cells suppresses endothelial cell growth and tumor formation ability of pancreatic cancers in vivo.Using nestin to more accurately evaluate microvessel density in cancer specimens may be a novel prognostic indicator.Furthermore,nestin-targeted therapy may suppress tumor proliferation via inhibition of angiogenesis in numerous malignancies,including pancreatic cancer.In this review article,we focus on nestin as a novel angiogenesis marker and possible therapeutic target via inhibition of tumor angiogenesis.     

siRNA targeting VEGF inhibits hepatocellular carcinoma growth and tumor angiogenesis in vivo

BACKGROUND/AIMS: We have investigated whether siRNA targeted against VEGF inhibits functional properties of endothelial cells in vitro and HCC tumor growth and blood vessel formation in vivo. METHODS: The influence of siRNA-VEGF on endothelial cell proliferation, apoptosis and tube formation were analyzed in vitro. Antitumoral effects were examined in an orthotopic tumor model after ex vivo transfer or intraperitoneal treatment of siRNA, respectively. Intratumoral microvessel density was assessed by CD31 staining. RESULTS: VEGF expression was inhibited in Hepa129 by 70% and in SVEC4-10 by 48% within two days after transfection. In vitro, endothelial cell proliferation and tube formation was reduced by 23% and 38%, respectively. Interference with VEGF signaling was demonstrated by reduced pAKT in hepatoma cells. Tumor growth was inhibited by ex vivo transfer or intraperitoneal application of siRNA-VEGF by 83% or 63% in orthotopic tumors within 14 days. VEGF protein was reduced in both models by 29% and 44%. Microvessel density dropped to 34% for tumors from ex vivo transfected cells and 39% for systemic treated tumors. CONCLUSIONS: The results show that VEGF knockdown can be associated with reduced endothelial cell proliferation and tube formation in vitro and decreased tumor growth and microvessel density in vivo.     

Xiaotan Sanjie decoction attenuates tumor angiogenesis by manipulating Notch-1-regulated proliferation of gastric cancer stem-like cells

AIM: To determine the underlying mechanisms of action and influence of Xiaotan Sanjie (XTSJ) decoction on gastric cancer stem-like cells (GCSCs).METHODS: The gastric cancer cell line MKN-45 line was selected and sorted by FACS using the cancer stem cell marker CD44; the stemness of these cells was checked in our previous study. In an in vitro study, the expression of Notch-1, Hes1, Vascular endothelial growth factor (VEGF), and Ki-67 in both CD44-positive gastric cancer stem-like cells (GCSCs) and CD44-negative cells was measured by Western blot. The effect of XTSJ serum on cell viability and on the above markers was measured by MTT assay and Western blot, respectively. In an in vivo study, the ability to induce angiogenesis and maintenance of GCSCs in CD44-positive-MKN-45- and CD44-negative-engrafted mice were detected by immunohistochemical staining using markers for CD34 and CD44, respectively. The role of XTSJ decoction in regulating the expression of Notch-1, Hes1, VEGF and Ki-67 was measured by Western blot and real-time polymerase chain reaction.RESULTS: CD44⁺ GCSCs showed more cell proliferation and VEGF secretion than CD44-negative cells in vitro, which were accompanied by the high expression of Notch-1 and Hes1 and positively associated with tumor growth (GCSCs vs CD44-negative cells, 2.72 ± 0.25 vs 1.46 ± 0.16, P < 0.05) and microvessel density (MVD) (GCSCs vs CD44-negative cells, 8.15 ± 0.42 vs 3.83 ± 0.49, P < 0.001) in vivo. XTSJ decoction inhibited the viability of both cell types in a dose-dependent manner in vitro. Specifically, a significant difference in the medium- (82.87% ± 6.53%) and high-dose XTSJ groups (77.43% ± 7.34%) was detected at 24 h in the CD44⁺ GCSCs group compared with the saline group (95.42% ± 5.76%) and the low-dose XTSJ group (90.74% ± 6.57%) (P < 0.05). However, the efficacy of XTSJ decoction was reduced in the CD44^- groups; significant differences were only detected in the high-dose XTSJ group at 48 h (78.57% ± 6.94%) and 72 h (72.12% ± 7.68%) when compared with the other CD44- groups (P < 0.05). Notably, these differences were highly consistent with the Notch-1, Hes1, VEGF and Ki-67 expression in these cells. Similarly, in vivo, XTSJ decoction inhibited tumor growth in a dose-dependent manner. A significant difference was observed in the medium- (1.76 ± 0.15) and high-dose XTSJ (1.33 ± 0.081) groups compared with the GCSCs control group (2.72 ± 0.25) and the low-dose XTSJ group (2.51 ± 0.25) (P < 0.05). We also detected a remarkable decrease of MVD in the medium- (7.10 ± 0.60) and high-dose XTSJ (5.99 ± 0.47) groups compared with the GCSC control group (8.15 ± 0.42) and the low-dose XTSJ group (8.14 ± 0.46) (P < 0.05). Additionally, CD44 expression was decreased in these groups [medium- (4.43 ± 0.45) and high-dose XTSJ groups (3.56 ± 0.31) vs the GCSC control (5.96 ± 0.46) and low dose XTSJ groups (5.91 ± 0.38)] (P < 0.05). The significant differences in Notch-1, Hes1, VEGF and Ki-67 expression highly mirrored the results of XTSJ decoction in inhibiting tumor growth, MVD and CD44 expression.CONCLUSION: Notch-1 may play an important role in regulating the proliferation of GCSCs; XTSJ decoction could attenuate tumor angiogenesis, at least partially, by inhibiting Notch-1.     

慢病毒Hoxa3载体的构建及其对人脐静脉内皮细胞迁移和血管新生的影响

目的构建慢病毒Hoxa3载体,观察其对人脐静脉内皮细胞(HUVEC)的转染效率,研究其对细胞迁移和血管新生的影响,并探讨Hoxa3促进血管新生的机制。方法以基因合成法从聚合酶链式反应文库获取人Hoxa3基因,酶切后插入慢病毒骨架载体,以三质粒联合转染293T细胞获得慢病毒Hoxa3载体,并进行滴度测定。转染HUVEC,获取最大转染效率。转染HUVEC后分对照组和慢病毒Hoxa3转染组,进行HUVEC迁移实验和小管形成实验,观察Hoxa3对HUVEC迁移和小管形成的影响。Western blot检测慢病毒Hoxa3载体转染HUVEC后尿激酶型纤溶酶原激活物受体(uPAR)和基质金属蛋白酶14(MMP-14)蛋白表达的变化。结果成功构建慢病毒Hoxa3载体,病毒的滴度为8×1011TU/L; 30 MOI慢病毒载体对HUVEC的转染效率达到99%以上。Western blot结果显示慢病毒Hoxa3载体转染HUVEC后Hoxa3能够有效在HUVEC中表达。与对照组比较,慢病毒Hoxa3载体转染HUVEC后显著增强HUVEC的迁移和小管形成,显著增加uPAR和MMP-14蛋白的表达(P0.05)。结论成功构建的慢病毒Hoxa3载体可促进HUVEC的迁移和小管形成,其作用机制可能为上调HUVEC的uPAR和MMP-14蛋白表达。     

The urokinase plasminogen activator system in cancer: Implications for tumor angiogenesis and metastasis

Substantial evidence exists which implicates the urokinase plasminogen activator system [urokinase plasminogen activator (uPA), urokinase plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1)] in the neo-vascularization, invasion and metastasis of many solid tumors. Clinical studies have demonstrated an association between high levels of expression of the components of this system in tumors and poor patient prognosis and outcome. Components of the uPA/uPAR system are differentially expressed or activated on motile cells including invading tumor cells and leukocytes, and migrating endothelial cells. In contrast, there is little or no expression on most normal, quiescent cells. Studies performed in vitro have demonstrated the regulation of the expression of uPA and uPAR by growth and differentiation factors as well as by oncogenes. In this review, we summarize recent findings on the role of the components of the uPA/uPAR system in angiogenesis, invasiveness and tumor metastasis. The activities of this system in endothelial and leukocyte cell biology and the relevance of these activities to angiogenesis and tumor metastasis will be considered. Recent experimental evidence obtained using inhibitors of uPA and uPAR has validated this system as a therapeutic target for the development of anti-angiogenic and anti-metastatic therapeutic agents. These studies, as well as additional therapeutic and diagnostic implications for uPAR targeting, will be discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

神经生长因子对大鼠心肌损伤后交感神经去支配及巢蛋白表达的影响

目的研究神经生长因子（NGF）预处理对异丙肾上腺素（Iso）致大鼠心肌急性损伤后交感神经去支配及巢蛋白（Nestin）表达的影响。方法雄性Wistar大鼠60只，随机分成3组：对照组、Iso损伤组和NGF预处理组．每组20只。观察心肌损伤的病理变化和血清肌酸激酶（CK）改变；免疫组化和RT～PCR方法检测心肌酪氨酸羟化酶（TH）和Nestin的表达。结果NGF预处理组较Iso损伤组心肌坏死面积明显减小（P〈0．05）。CK值在Iso损伤组最高，NGF预处理组次之，均高于对照组（P〈0．01）。TH染色阳性的交感神经纤维密度在Iso损伤组[（20．63±6．04）个／mm^2]明显下降，与对照组[（31．40±8．96）个／mm^2]比较，差异有统计学意义（P〈0．05），而NGF预处理组[（29．22±9．11）个／mm^2]TH阳性表达与对照组水平接近，并未发生显著减少。各组THmRNA表达水平与免疫组化结果平行。对照组NestinmRNA表达水平最低，Iso损伤组和NGF预处理组较高，以NGF预处理组为著。结论NGF预处理可拮抗Iso致大鼠心肌损伤的，心脏交感神经去支配，减轻Iso的心肌损伤作用，提示NGF可通过神经营养作用在心肌损伤修复过程中起重要作用。     

结直肠癌干细胞研究进展

结直肠癌与其他实体肿瘤一样是我国乃至世界范围的重要疾病负担,其治疗也面临许多挑战.近几年,日益受到重视的癌症干细胞理论似乎可以解释结直肠癌的发生发展、复发转移的细胞学机制.本文就结直肠癌干细胞的研究进展作一综述.     

沙利度胺对人脐静脉内皮细胞增殖和血管生成的影响

目的 探讨沙利度胺治疗血管发育不良所致消化道出血的机制.方法 体外培养人脐静脉内皮细胞至对数生长期,分为空白对照组、溶剂对照组(二甲基亚砜)和不同浓度(10、20、40、60、80、100μg/ml)沙利度胺组,根据加或不加成纤维细胞生长因子(bFGF,10 ng/ml),共分为16组.刺激72 h后,MTT法检测细胞增殖情况,酶联免疫吸附法和实时定量PCR法测定血管内皮生长因子(VEGF)、肿瘤坏死因子-α(TNF-α)表达.结果 加或不加bFGF刺激,中、高浓度(≥40/μg/ml)沙利度胺均能抑制人脐静脉内皮细胞增殖.未加bFGF刺激时.20μg/ml沙利度胺能明显抑制VEGF表达.加bFGF刺激时,10 μg/ml沙利度胺即能明显抑制VEGF表达.未检出TNF-α表达.结论 体外实验中,沙利度胺能抑制人脐静脉内皮细胞增殖和VEGF表达,从而抑制血管生成,达到治疗血管发育不良所致消化道出血的目的 .     

A hybrid protein of the amino-terminal fragment of urokinase and mutant plasminogen activator inhibitor-2 efficiently inhibits tumor cell invasion and metastasis

The urokinase plasminogen activator(uPA) system plays important roles in tumor cell invasion and metastasis. In the present study, we evaluated the effects of ATF-PAI2CD, a hybrid protein of the amino-terminal fragment of urokinase and mutant plasminogen activator inhibitor-2, on 95D cells in vitro and in vivo. Furthermore, our results support a current hypothesis that fusion protein blocks tumor invasion and motility by inhibiting localized pericellular proteolysis. Treatment of 95D cells with ATF-PAI2CD resulted in a dose-dependent decrease in tumor-cell invasion through matrigel, and ATF-PAI2CD was much more effective than PAI-2CD. In addition, extracellular regular protein kinase (ERK1/2) expression was downregulated and the adhesion ability to fibronectin was increased in 95D cells treated with the fusion protein, which was confirmed by cell adhesion assay. A high-concentration of ATF-PAI2CD caused a significant reduction in tumor volume and weight in BALB/c (nu/nu) mice female inoculated with human 95D cells (5×10⁶); the antitumor effects were significant, which demonstrated a 67.9±4.2% reduction in tumor growth compared with control mice. The number of lymphatic metastasis was significantly reduced in mice treated with high- and middle- concentrations of ATF-PAI2CD, whereas a low-concentration of ATF-PAI2CD failed to exhibit any antimetastatic effects. In conclusion, the results suggested that the hybrid protein has therapeutic potential for lung carcinoma and other tumors to inhibit tumor invasion and metastasis.     

Effect of Src kinase inhibition on metastasis and tumor angiogenesis in human pancreatic cancer

Tumor angiogenesis is a process that requires migration, proliferation, and differentiation of endothelial cells. We hypothesized that decrease in pancreatic tumor growth due to inhibition of Src activity is associated with the inability of Src kinase to trigger a network of such signaling processes, which finally leads to endothelial cell death and angiogenesis-restricted tumor dormancy. The therapeutic efficacy of Src kinase inhibitor AZM475271 was tested in nude mice orthotopically xenografted with L3.6pl pancreatic carcinoma cells. No liver metastases and peritoneal carcinosis were detected and a significant effect on the average pancreatic tumor burden was observed following treatment with AZM475271, which in turn correlated with a decrease in cell proliferation and an increase in apoptotic endothelial cells. AZM475271 was shown to significantly inhibit migration of human umbilical vein endothelial cells in an in vitro Boyden Chamber cell migration assay. In a rat aortic ring assay we could demonstrate as well inhibition of endothelial cell migration and sprouting following therapy with Src kinase inhibitor at similar doses. The most conclusive anti-angiogenic activity of AZM475271 was demonstrated in vivo (mouse corneal micropocket assay) by showing a marked inhibition of basic fibroblast growth factor-induced neovascularization in response to systemic administration of AZM475271. Furthermore, we could show reduced proliferation of HUVECs determined with the TACS MTT Cell Viability Assay Kit. The blockade of Src kinase significantly reduced the level of VEGF in L3.6pl medium, the effect which was found also in the cell culture supernate from HUVECs. Inhibition of Src kinase by AZM475271 also showed prevention of survival signaling from VEGF and EGF receptors. Treatment with AZM475271 resulted in VEGF - dependent inhibition of tyrosine phosphorylation of FAK. HUVECs were also examined using propidium iodide staining for cell cycle analysis by FACS. Inhibition of Src kinase promoted HUVEC apoptosis in a dose-dependent manner. Taken together, our results suggest that the Src kinase inhibitor AZM475271, in addition to its effects on tumor cells, suppresses tumor growth and metastasis in vitro and in vivo potentially also by anti-angiogenic mechanisms.     

Hallmarks in colorectal cancer:Angiogenesis and cancer stem-like cells

Carcinogenesis is a multistep process that requires the accumulation of various genetic and epigenetic aberrations to drive the progressive malignant transformation of normal human cells.Two major hallmarks of carcinogenesis that have been described are angiogenesis and the stem cell characteristic of limitless replicative potential.These properties have been targeted over the past decade in the development of therapeutic treatments for colorectal cancer(CRC),one of the most commonly diagnosed and lethal cancers worldwide.The treatment of solid tumor cancers such as CRC has been challenging due to the heterogeneity of the tumor itself and the chemoresistance of the malignant cells.Furthermore,the same microenvironment that maintains the pool of intestinal stem cells that contribute to the continuous renewal of the intestinal epithelia also provides the necessary conditions for proliferative growth of cancer stem-like cells.These cancer stem-like cells are responsible for the resistance to therapy and cancer recurrence,though they represent less than 2.5%of the tumor mass.The stromal environment surrounding the tumor cells,referred to as the tumor niche,also supports angiogenesis,which supplies the oxygen and nutrients needed for tumor development.Anti-angiogenic therapy,such as with bevacizumab,a monoclonal antibody against vascular-endothelial growth factor,significantly prolongs the survival of metastatic CRC patients.However,such treatments are not completely curative,and a large proportion of patient tumors retain chemoresistance or show recurrence.This article reviews the current knowledge regarding the molecular phenotype of CRC cancer cells,as well as discusses the mechanisms contributing to their maintenance.Future personalized therapeutic approaches that are based on the interaction of the carcinogenic hallmarks,namely angiogenic and proliferative attributes,could improve survival and decrease adverse effects induced by unnecessary chemotherapy.     

Bone marrow angiogenesis and circulating plasma cells in multiple myeloma

Bone marrow (BM) angiogenesis is increased in multiple myeloma (MM) and has prognostic significance. The presence of circulating plasma cells (PCs) in MM is associated with a poorer prognosis. We examined BM biopsies obtained at diagnosis of MM for angiogenesis, and correlated the microvessel density (MVD) with the presence of circulating PCs. There was a positive correlation between the absolute number of circulating PCs and the mean MVD. This relationship was independent of the disease activity and of the PC burden in the marrow. The increased angiogenesis may promote plasma cell proliferation and enable PC migration into the circulation.     

Netrins and UNC5 receptors in angiogenesis

Both neuronal and vascular development require guidance to establish a precise branching pattern of these systems in the vertebrate body. Several molecules implicated in axon navigation have also been shown to regulate vessel sprouting. Among these guidance cues, Netrins constitute a family of diffusible molecules with a bifuncional role in axon pathfinding. Recent findings implicate Netrins in other developmental processes, including vascular development. We here review recent studies and discuss the possible dual function of Netrins and its receptors during branching of blood vessels in developmental and pathological angiogenesis.     

清脑益元汤对大鼠脑缺血损伤Nestin、NGF表达的影响

目的研究清脑益元汤对大鼠脑缺血损伤后巢蛋白(Nestin)、神经生长因子(NGF)表达的影响,探究清脑益元汤对缺血性脑损伤神经元保护作用的部分机制。方法将192只健康SD大鼠随机分为两大组,每大组再分为4个亚组:空白组(n=6)、假手术组(n=30)、模型组(n=30)、清脑益元汤组(n=30)。采用改良的Longa线栓法制备大鼠急性脑缺血损伤模型,除空白组外,假手术组、模型组及清脑益元组按缺血损伤后1 d、3 d、7 d、14 d、28 d 5个取材时间点分为5个亚组。造模成功后取大鼠缺血侧脑组织,采用免疫组化法检测大鼠缺血侧皮质区Nestin、NGF表达,采用实时荧光定量PCR(Real-Time PCR)法检测大鼠缺血侧皮质区Nestin、NGF mRNA表达。结果(1)免疫组化法:与假手术组相比,模型组、清脑益元组在缺血侧Nestin、NGF阳性细胞明显增多(P<0.01,P<0.01);与模型组相比,清脑益元组Nestin、NGF阳性细胞表达明显增多(P<0.01,P<0.05);(2)Real-Time PCR法:与假手术组相比,模型组、清脑益元汤组大鼠脑组织缺血侧皮质区Nestin、NGF mRNA表达量增加(P<0.01,P<0.01);与模型组相比,清脑益元汤组大鼠脑组织缺血侧皮质区Nestin、NGF mRNA表达量升高(P<0.01,P<0.05)。结论清脑益元汤可能通过上调脑缺血损伤后Nestin、NGF蛋白及mRNA表达,促进脑组织损伤修复、保护神经元,进而发挥脑保护的作用。     

The biology of cancer stem cells and its clinical implication in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a highly malignant tumor with limited treatment options in its advanced state. The molecular mechanisms underlying HCC remain unclear because of the complexity of its multi-step development process. Cancer stem cells (CSCs) are defined as a small population of cells within a tumor that possess the capability for self-renewal and the generation of heterogeneous lineages of cancer cells. To date, there have been two theories concerning the mechanism of carcinogenesis, i.e., the stochastic (clonal evolution) model and the hierarchical (cancer stem cell-driven) model. The concept of the CSC has been established over the past decade, and the roles of CSCs in the carcinogenic processes of various cancers, including HCC, have been emphasized. Previous experimental and clinical evidence indicated the existence of liver CSCs; however, the potential mechanistic links between liver CSCs and the development of HCC in humans are not fully understood. Although definitive cell surface markers for liver CSCs have not yet been found, several putative markers have been identified, which allow the prospective isolation of CSCs from HCC. The identification and characterization of CSCs in HCC is essential for a better understanding of tumor initiation or progression in relation to signaling pathways. These markers could be used along with clinical parameters for the prediction of chemoresistance, radioresistance, metastasis and survival and may represent potential targets for the development of new molecular therapies against HCC. This review describes the current evidence for the existence and function of liver CSCs and discuss the clinical implications of CSCs in patients demonstrating resistance to conventional anti-cancer therapies, as well as clinical outcomes. Such data may provide a future perspective for targeted therapy in HCC.     

miRNA与肿瘤干细胞的研究进展

微小RNA（miRNA）是一类参与转录后调控的单链非编码小分子RNA,能够调节多种病理生理过程,如干细胞分化、细胞增殖和肿瘤形成,并且某些miRNA与肿瘤干细胞的自我更新和多向分化存在紧密联系。因此,miRNA作为一种新的调控基因表达的小分子RNA,可能成为研究肿瘤于细胞的新途径。