Differential Effects of Thalidomide on Angiogenesis and Tumor Growth in Mice

Thalidomide, clinically used as an antiinflammatory and antitumoral drug, inhibited sponge-induced angiogenesis when administered systemically (100 mg/kg^–1) in mice. However, it failed to inhibit solid Ehrlich tumor in the same mouse strain. We have used functional, biochemical and histological parameters to assess neovascularization and fibrovascular tissue infiltration of the mice sponge granuloma. The neovascularization growth as detected by development of blood flow and hemoglobin content extracted from the implants showed that thalidomide inhibited fibrovascular tissue formation by 40%. The functional and biochemical parameters correlated well with the histological study. Thalidomide had no inhibitory effect in the development of Ehrlich tumor. The detection of this selective action using the same animal strain bearing two different processes, supports the hypothesis that rather than species specificity, thalidomide is tissue specific. This approach may be used to identify the specificity of other therapeutic agents against distinct angiogenesis-dependent diseases.     

Upregulation of vascular endothelial growth factor by cobalt chloride-simulated hypoxia is mediated by persistent induction of cyclooxygenase-2 in a metastatic human prostate cancer cell line

Upregulation of vascular endothelial growth factor (VEGF) expression induced by hypoxia is crucial event leading to neovascularization. Cyclooxygenase-2, an inducible enzyme that catalyzes the formation of prostaglandins (PGs) from arachidonic acid, has been demonstrated to be induced by hypoxia and play role in angiogenesis and metastasis. To investigate the potential effect of COX-2 on hypoxia-induced VEGF expression in prostate cancer. We examined the relationship between COX-2 expression and VEGF induction in response to cobalt chloride (CoCl₂)-simulated hypoxia in three human prostate cancer cell lines with differing biological phenotypes. Northern blotting and ELISA revealed that all three tested cell lines constitutively expressed VEGF mRNA, and secreted VEGF protein to different degrees (LNCaP > PC-3 > PC3ML). However, these cell lines differed in the ability to produce VEGF in the presence of CoCl₂-simulated hypoxia. CoCl₂ treatment resulted in 40% and 75% increases in VEGF mRNA, and 50% and 95% in protein secretion by LNCaP and PC-3 cell lines, respectively. In contrast, PC-3ML cell line, a PC-3 subline with highly invasive, metastatic phenotype, exhibits a dramatic upregulation of VEGF, 5.6-fold in mRNA and 6.3-fold in protein secretion after treatment with CoCl₂. The upregulation of VEGF in PC-3ML cells is accompanied by a persistent induction of COX-2 mRNA (6.5-fold) and protein (5-fold). Whereas COX-2 expression is only transiently induced in PC-3 cells and not affected by CoCl₂ in LNCaP cells. Moreover, the increases in VEGF mRNA and protein secretion induced by CoCl₂ in PC-3ML cells were significantly suppressed following exposure to NS398, a selective COX-2 inhibitor. Finally, the effect of COX-2 inhibition on CoCl₂-induced VEGF production was reversed by the treatment with exogenous PGE₂. Our data demonstrate that VEGF induction by cobalt chloride-simulated hypoxia is maintained by a concomitant, persistent induction of COX-2 expression and sustained elevation of PGE₂ synthesis in a human metastatic prostate cancer cell line, and suggest that COX-2 activity, reflected by PGE₂ production, is involved in hypoxia-induced VEGF expression, and thus, modulates prostatic tumor angiogenesis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Differential assessment of angiogenic activity and of vascular survival ability (VSA) in breast cancer

Recent reports provide evidence that some growth factors behave as inhibitors of the apoptosis of the endothelial cells, bringing forward the concept of vascular survival as a post-angiogenesis process. At least two different vasculature development processes occur within a tumor: the angiogenic (formation of new vessels) and the vascular survival pathway, which is devoted to the preservation of the newly-formed vessels in layers that lose contact with the adjacent normal tissue. We developed a method to assess these processes in tissue samples. We noted that differences among tumors may exist not only in the tumor angiogenic activity (TAA) but also in the vascular survival ability (VSA). One third of the highly angiogenic breast cancer cases examined had a poor ability to maintain high vessel density in inner tumor areas. Both parameters are independently related to prognosis, while VSA was directly related to tumor dimensions and node involvement. Patients with high TAA and VSA had a particularly poor prognosis. It is suggested that although cancer angiogenic activity is important for the local invasion and dissemination into vessels and lymphatics, the VSA may be important for the effective formation of viable tumor foci in lymph nodes or distant organs. Recognition and quantification of the vascular survival ability in human tumors may significantly improve the prognostic value of the assessment of tumor vasculature, and may help to stratify patients for clinical trials with novel anti-angiogenic or angiotoxic drugs. Elucidation of the pathways may provide additional targets for antiangiogenic therapy. This revised version was published online in July 2006 with corrections to the Cover Date.     

The relationship of VEGF and PGE2 expression to extracellular matrix remodelling of the tenosynovium in the carpal tunnel syndrome

Tenosynovial thickening within the confined space of the carpal tunnel is thought to be the cause of the carpal tunnel syndrome (CTS). However, little is known about the pathological mechanism of tenosynovial thickening. In this study, the role of prostaglandin E(2) (PGE(2)) and vascular endothelial growth factor (VEGF) (two representative molecules that can induce oedema by increasing vascular permeability) was analysed in CTS by using immunohistochemistry and enzyme-linked immunosorptive assay (ELISA). Expression of these molecules was compared with the patients' clinical histories and a temporary increase in production of these molecules was found in cells within the vessels and synovial lining during the intermediate phase of the syndrome when the histology of the tenosynovium changes from oedematous to fibrotic. Statistical analysis clearly demonstrated that there is a close correlation between the expression of PGE(2) and VEGF. Furthermore, immunohistochemical analysis with anti-proliferating cell nuclear antigen (PCNA) revealed that the area with distinct VEGF expression closely matched the area where endothelial cells, vascular smooth muscle cells, and synovial lining cells proliferate. In contrast, despite marked alteration in the extracellular matrix (ECM) component of the tenosynovium, the fibroblasts responsible for most ECM framework production do not proliferate during any phase of CTS. Histological analysis demonstrated that angiogenesis takes place only during the intermediate phase. Since clusters of capillaries and arterioles are often surrounded by type III collagen-rich, disorganized, degenerate connective tissue, which contains fewer fibroblasts than normal, angiogenesis appears to take place as a part of a regenerative reaction that results in fibrosis. These findings strongly indicate that both PGE(2) and VEGF are expressed in the tenosynovium in CTS during the intermediate phase and induce the histological changes seen in the tenosynovium.     

Erythropoietin concentrations are elevated in the peritoneal fluid of women with endometriosis

Erythropoietin (Epo) is an important regulator of erythropoiesis and stimulates the proliferation of early erythroid precursors as well as the differentiation of late erythroid precursors of the erythroid lineage. However, recent studies have indicated that Epo also has angiogenic properties and plays an important role in the oestrogen-dependent cyclical angiogenesis within the mouse uterus. It was therefore postulated that Epo may be an important angiogenic factor in endometriosis. In order to address this hypothesis the concentration of Epo in peritoneal fluid (PF) was determined in patients with or without endometriosis. PF was collected from patients with endometriosis (n = 42) or without endometriosis (n = 18). Detectable concentrations of Epo were found in all PF samples analysed. The concentration of Epo in PF from patients with endometriosis was significantly higher than that in the control group (13.1 +/- 1.2 mIU/ml versus 7.2 +/- 0.7 mIU/ml, mean +/- SE respectively, P < 0.01). Furthermore, in patients with endometriosis the Epo concentrations in PF from patients with stage I disease (n = 17, 16.6 +/- 3.0 mIU/ml) were significantly higher than those with stage II (n = 8, 10.7 +/- 1.2 mIU/ml, P < 0.03), III (n = 13, 8.4 +/- 1.0 mIU/ml, P < 0.01), IV disease (n = 7, 7.5 +/- 1.0 mIU/ml, P < 0.01). These data suggest that Epo may play a role in the pathogenesis of endometriosis particularly in the initiation of the disease.     

碱性成纤维细胞生长因子与卵巢癌的关系

目的　探讨碱性成纤维细胞生长因子 (basic fibroblast growth factor,b FGF)对卵巢癌细胞增殖、浸润和肿瘤血管生成的影响 ,及 b FGF单克隆抗体 (b FGF monoclonal antibody,b FGF- MAb)的治疗作用。　方法　将人卵巢癌细胞株 SKOV3接种于 2 4孔板 ,加入不同浓度的 b FGF,每日行结晶紫染色后测定光密度 (D4 90 )值 ,绘制细胞生长曲线 ;将 SKOV3细胞团接种于铺设有细胞外基质凝胶的 4孔板 ,每日测定癌细胞在凝胶中的浸润距离 ;建立 SKOV3细胞裸鼠皮下移植瘤模型 ,每周两次分别将 b FGF、b FGF-MAb和生理盐水注射于移植瘤周围 ,8周后测量肿瘤体积 ;对移植瘤组织切片行 因子的免疫组化染色、测定肿瘤内微血管密度 (microvessel density,MVD)。　结果　 b FGF能促进 SKOV3细胞增殖并呈浓度依赖 ,实验第 5天 ,5 ng/ml、10 ng/ml组细胞 D4 90 值是对照组的 1.0 9倍和 1.2 1倍 ;b FGF能促进 SKOV3细胞浸润并呈浓度依赖 (P<0 .0 5 ) ,第 7天 ,5 ng/ml、10 ng/ml组细胞浸润距离分别是对照组的 1.5 3倍和2 .4 5倍 ;b FGF组移植瘤体积和 MVD分别是对照组的 1.80倍和 1.4 6倍 (P<0 .0 5 ) ,b FGF- MAb组移植瘤体积和 MVD分别是对照组的 6 3.7%和 6 2 .8% (P<0 .0 5 )。　结论　b FGF能明显促进卵巢癌细胞的增殖、     

The expression patterns of mRNA-encoding stem cell factor, internal stem cell factor and c-kit in the prepubertal and adult porcine ovary

The receptor, c-kit, and its ligand, stem cell factor (SCF), are important regulators of ovarian follicle growth and development. The aim of this study was to identify the sites of expression of mRNA for c-kit and SCF in prepubertal and mature (pregnant and non-pregnant) animals. Ovaries were recovered from prepubertal animals, non-pregnant sows and five sows at approximately 3 months of gestation. Ovine SCF and c-kit DNA were cloned into plasmid vectors to produce RNA probes. Expression of mRNA encoding SCF and c-kit were detected via in situ hybridization. Both mRNA were detected throughout ovaries from all animals. This study provides evidence that the growth-factor complex is required throughout follicle development, and also for continued maintenance of the corpus luteum (CL) in the mature animal. SCF mRNA was localized to the granulosa cell layer and was also extensively expressed in endothelial tissue and throughout the CL. c-kit mRNA was detected in the theca layer, oocytes and also in CL. In conclusion, expression of SCF and c-kit mRNA in granulosa and theca cells, respectively, indicate an important interaction between somatic cells throughout follicle development and that in the mature animal, SCF and c-kit potentially have a role in maintaining progesterone secretion by the CL. The observations of continued expression of SCF and c-kit throughout development suggest that there may be differences in the role of this receptor-ligand complex between large mono- vs. poly ovulatory species, such as the pig.     

Phosphorylated KDR is expressed in the neoplastic and stromal elements of human renal tumours and shuttles from cell membrane to nucleus

Vascular endothelial growth factor (VEGF)-A is an important angiogenic factor in establishing the vasculature in renal cell carcinomas (RCCs). Since little is known about VEGF signalling in RCCs, the profile of phosphorylated KDR (pKDR) has been investigated and the intracellular location of the receptor has been examined in the present study. Using two monoclonal antibodies raised against the phosphorylated KDR epitopes (Y1059 and Y1214) known to mediate different VEGF functions, together with a commercial anti-KDR antibody and immunohistochemistry, the expression of pKDR was investigated in a series of normal (n = 25) and neoplastic kidneys (n = 54; clear cell n = 35; papillary n = 10; oncocytomas n = 8). pKDR was present in many tissue elements of both normal and neoplastic renal tissues, with strong expression in the cell membrane, cytoplasm, and nuclei of normal kidney and tumour cells, as well as endothelial cells in tumours of all histological types. Patterns and intensity were similar using both anti-pKDR antibodies. There was no significant correlation in clear cell carcinomas between pKDR expression and age (p = 0.57), tumour size (p = 0.2), gender (p = 0.59), grade (p = 0.2) or histological type (p = 0.36). To delineate further the intracellular processing that might account for the cellular distribution, confocal microscopy was also performed. Antibodies to the different phosphorylated epitopes demonstrated different intracellular staining patterns. This study shows that pKDR is present in a wide variety of renal tumours, suggesting that anti-VEGF therapy might have direct effects on tumour cells. It further suggests that cells traffic pKDR depending on the precise KDR tyrosines that are autophosphorylated in a manner that enables receptor activation to result in different functions.     

Familial prevalence of uterine fibroids is associated with distinct clinical and molecular features

BACKGROUND: Although uterine fibroids are very common, their pathogenesis and clinical behaviour are poorly understood. Since they may be prevalent in some families, we investigated whether such a prevalence was associated with distinctive clinical and molecular features. METHODS: A case-control questionnaire study of 300 multi-ethnic women with uterine fibroids at a London university hospital was undertaken, with review of case notes and immunohistochemical determination of vascular endothelial growth factor (VEGF-A) in fibroids. RESULTS: When compared with families with sporadic fibroids, familial prevalence of fibroids was associated with a higher incidence of abdominal swelling (59.1% versus 41.6%; P=0.037), menorrhagia (84.4% versus 51.9%; P=0.042), dysmenorrhoea (64.4% versus 46.3%; P=0.004), dyspareunia (43.2% versus 27.9%; P=0.012) and family history of cancers (52.3% versus 32.4%; P<0.01). The fibroids were also more multiple (mean +/- SEM: 7 +/- 0.86 versus 3 +/- 0.42; P<0.011) and strong VEGF-A expression in fibroids was more common in the familial group (64% versus 28%). Racial distribution was the same in both groups (blacks 49%, whites 33.4%, others 18.6%). CONCLUSIONS: Familial prevalence of uterine fibroids is associated with distinct clinical and molecular features that differ from those found when fibroids occur sporadically in families.     

经冠脉移植骨髓单个核细胞对猪梗死心肌的修复作用

目的观察经冠脉移植骨髓单个核细胞的定植、分化和对心功能的影响。方法小型猪冠状动脉前降支结扎90min后再灌注制备心肌梗死模型,分为骨髓细胞移植组(BM-MNCs组,n=6)和对照组(n=5),分别经冠脉移植PKH26标记的骨髓单个核细胞和培养基,术后6周进行病理学检查并行血流动力学和超声心动图检查心功能变化。结果在BM-MNCs组,心肌梗死后6周在缺血心肌内可找到移植细胞,其Ⅷ因子和desmin免疫组化染色均为阳性;血流动力学指标显示左室等容舒张期压力最大变化率和心排量与心肌梗死后90min比较明显改善(P<0.05);超声心动图显示每搏输出量和心排量比对照组明显改善(P<0.05);心肌梗死边缘区的小血管数目明显多于对照组(P<0.05)。结论经冠脉移植骨髓单个核细胞可定植于梗死区心肌,并可分化为肌源性细胞和血管内皮细胞,可促进小血管再生,有改善心功能的潜能。