Thrombomodulin is a determinant of metastasis through a mechanism linked to the thrombin binding domain but not the lectin-like domain

Thrombomodulin (TM) is a predominantly endothelial transmembrane glycoprotein that modulates hemostatic function through a domain that controls thrombin-mediated proteolysis and an N-terminal lectin-like domain that controls inflammatory processes. To test the hypothesis that TM is a determinant of malignancy and dissect the importance of these functional domains in cancer biology, metastatic potential was evaluated in TM(Pro) mice expressing a mutant form of TM with reduced thrombin affinity and TM(LeD) mice lacking the N-terminal lectin-like domain. Studies of TM(Pro) mice revealed that TM is a powerful determinant of hematogenous metastasis. TM(Pro) mice exhibited a strongly prometastatic phenotype relative to control mice that was found to result from increased survival of tumor cells newly localized to the lung rather than any alteration in tumor growth. The impact of the TM(Pro) mutation on metastasis was dependent on both tumor cell-associated tissue factor and thrombin procoagulant function. In contrast, expression of a mutant form of TM lacking the lectin-like domain had no significant impact on metastasis. These studies directly demonstrate for the first time that TM-mediated regulation of tumor cell-driven procoagulant function strongly influences metastatic potential and suggest that endothelial cell-associated modulators of hemostasis may represent novel therapeutic targets in limiting tumor dissemination.     

Plasminogen is a critical determinant of vascular remodeling in mice

Extracellular proteolysis is likely to be a feature of vascular remodeling associated with atherosclerotic and restenotic arteries. To investigate the role of plasminogen-mediated proteolysis in remodeling, polyethylene cuffs were placed around the femoral arteries of mice with single and combined deficiencies in plasminogen and fibrinogen. Neointimal development occurred in all mice and was unaffected by genotype. Significant compensatory medial remodeling occurred in the cuffed arteries of control mice but not in plasminogen-deficient mice. Furthermore, focal areas of medial atrophy were frequently observed in plasminogen-deficient mice but not in control animals. A simultaneous deficit of fibrinogen restored the potential of the arteries of plasminogen-deficient mice to enlarge in association with neointimal development but did not eliminate the focal medial atrophy. An intense inflammatory infiltrate occurred in the adventitia of cuffed arteries, which was associated with enhanced matrix deposition. Adventitial collagen deposition was apparent after 28 days in control and fibrinogen-deficient arteries but not in plasminogen-deficient arteries, which contained persistent fibrin. These studies demonstrate that plasmin(ogen) contributes to favorable arterial remodeling and adventitial collagen deposition via a mechanism that is related to fibrinogen, presumably fibrinolysis. In addition, these studies reveal a fibrin-independent role of plasminogen in preventing medial atrophy in challenged vessels.     

Disruption of hepatocellular tight junctions by vascular endothelial growth factor (VEGF): a novel mechanism for tumor invasion

BACKGROUND/AIMS: Vascular endothelial growth factor (VEGF) is expressed by many tumors, including hepatocellular carcinoma (HCC) and is involved in tumor angiogenesis. Little is known about its role for HCC infiltration into normal liver parenchyma. METHODS: The effects of VEGF on the integrity of tight junctions were studied in HepG2 cells and human HCC by means of confocal laser scanning microscopy. RESULTS: VEGF induced within 45 min a marked loss of pseudocanaliculi and disruption of occludin-delineated tight junctions. This effect of VEGF was mimicked by phorbol-12-myristate-13-acetate (PMA) and was sensitive to protein kinase C (PKC) inhibition by G?6850. VEGF induced within 15 min the translocation of the PKC alpha-isoform to the plasma-membrane, but had no effect on the activity of Erks and p38(MAPK). Sections from surgically removed HCC showed expression of VEGF in the tumor and occludin disassembly in normal liver parenchyma next to the tumor. CONCLUSIONS: VEGF induces disruption of tight junctions in a PKC-alpha dependent manner. In addition to its known angioneogenic properties, VEGF may promote HCC spreading into normal liver parenchyma. The data may provide another rationale for the use of VEGF antagonists for tumor therapy.     

Vascular endothelial growth factor induces SHC association with vascular endothelial cadherin: a potential feedback mechanism to control vascular endothelial growth factor receptor-2 signaling

Vascular endothelial (VE)-cadherin is endothelium specific, mediates homophilic adhesion, and is clustered at intercellular junctions. VE-cadherin is required for normal development of the vasculature in the embryo and for angiogenesis in the adult. Here, we report that VE-cadherin is associated with VE growth factor (VEGF) receptor-2 (VEGFR-2) on the exposure of endothelial cells to VEGF. The binding parallels receptor phosphorylation on tyrosine residues, which is maximal at 5 minutes and then declines within 30 minutes. Tyrosine phosphorylation of VE-cadherin was maximal at 30 minutes after the addition of the growth factor. At this time point, the protein could be coimmunoprecipitated with the adaptor protein Shc. Pull-down experiments with different Shc domains and mutants of the VE-cadherin cytoplasmic tail have shown that Shc binds to the carboxy-terminal domain of the VE-cadherin tail through its Src homology 2 domain (SH2). We found that Shc phosphorylation lasts longer in endothelial cells carrying a targeted null mutation in the VE-cadherin gene than in VE-cadherin-positive cells. These data suggest that VE-cadherin expression exerts a negative effect on Shc phosphorylation by VEGFR-2. We speculate that VE-cadherin binding to Shc promotes its dephosphorylation through associated phosphatases.     

Interleukin-17 inhibits tumor cell growth by means of a T-cell-dependent mechanism

Interleukin 17 (IL-17) is a proinflammatory cytokine produced by activated CD4(+) memory T cells. We previously showed that IL-17 increased the growth rate of human cervical tumors transplanted into athymic nude mice. To address the possible role of T cells in the biologic activity of IL-17 for tumor control, we grafted 2 murine hematopoietic immunogenic tumors (P815 and J558L) transfected with a complementary DNA encoding murine IL-17 into syngeneic immunocompetent mice. We found that growth of the 2 IL-17-producing tumors was significantly inhibited compared with that of mock-transfected tumors. In contrast to the antitumor activity of IL-17 observed in immunocompetent mice, we observed no difference in the in vivo growth of IL-17-transfected or mock-transfected P815 cells (P815-IL-17 and P815-Neo, respectively) transplanted into nude mice. We then showed that IL-17 increased generation of specific cytolytic T lymphocytes (CTLs) directed against the immunodominant antigens from P815 called A, B, C, D, and E, since all mice injected with P815-IL-17 developed a P815-specific CTL response, whereas only 6 of 16 mice immunized with P815-Neo had a specific CTL response against the antigens. The induction of CTLs was associated with establishment of a tumor-protective immunity. These experiments suggest that T lymphocytes are involved in the antitumor activity of IL-17. Therefore, IL-17, like other cytokines, appears to be a pleiotropic cytokine with possible protumor or antitumor effects on tumor development, which often depends on the immunogenicity of tumor models.     

Uric acid is closely linked to vascular nitric oxide activity : Evidence for mechanism of association with cardiovascular disease

OBJECTIVES

The study was undertaken to determine whether the mechanism of association of elevated serum uric acid level (SUA) with cardiovascular disease (CVD) is secondary to a common link with vascular nitric oxide (NO) activity.

BACKGROUND

Epidemiologic studies demonstrate an association of elevated SUA with CVD. The mechanism of this association is unknown, but both may be linked via an impairment in vascular NO activity. To examine this, we determined the relationship of SUA to vascular NO activity and to CVD risk. We then determined the effect of enhancing vascular NO activity on SUA.

METHODS

In part 1, individuals with various degrees of CVD (n = 458) were surveyed and underwent measurement of flow-mediated brachial artery vasodilation (FMV), a measure of vascular NO activity. In part 2, we performed an analysis of data pooled from six separate clinical trials of a medical food designed to enhance vascular NO activity in individuals with CVD (n = 217 subjects representing 253 treatment periods) to determine the effect on SUA.

RESULTS

In part 1, of all risk factors tested, SUA was second only to age in correlation with FMV, accounting for 7% (p < 0.0001) of the variability in FMV. Both SUA and FMV were related to the degree of disease risk (p < 0.0001 and P = 0.00025 by analysis of variance, respectively). By multivariate analysis, SUA did not continue to contribute significantly to the determination of FMV. In part 2, enhancement of FMV (5.8 ± 4 to 8.6 ± 5, p < 0.0001) was associated with a decrease in SUA (5.5 ± 1.5 to 5.0 ± 1.5, p < 0.0001). There was no placebo effect on FMV or SUA.

CONCLUSIONS

These results suggest that the association of elevated SUA with CVD may be a consequence of an impairment of vascular NO activity. This may be owing to an ability of NO to modulate uric acid production through its influence on xanthine oxidase activity.     

Plasminogen activator inhibitor 1, fibrin, and the vascular response to injury

Intravascular fibrin deposition is believed to play an important role in the development of intimal hyperplasia, which is a hallmark of several human vascular disorders, including atherosclerosis and restenosis after balloon angioplasty. Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor or tissue- and urinary-type plasminogen activator, plays a key role in fibrin homeostasis by controlling plasmin formation. PAI-1 may also modulate vascular pathology via alternative pathways, such as inhibiting activated protein C and altering interactions between vascular smooth muscle cells and the extracellular matrix. The diverse functional profile of PAI-1 likely accounts for the variation observed in its impact on intimal hyperplasia in different disease models. This review examines recent studies addressing the vascular function of PAI-1, and those assessing the role of fibrin as a downstream mediator of PAI-1's effects.     

IL-17A通过增强肿瘤血管生成和侵袭促进肿瘤发展

目的探讨IL-17A在非小细胞肺癌血管形成和侵袭中的作用。方法Lewis肺癌细胞（LLC）在不同浓度IL-17A（0、25、50、100和200μg／L）条件下行增殖实验,LLC在50μg／L IL-17A和0μg／LIL-17A刺激下行侵袭实验。LLC在IL-17A刺激不同时间点下用实时定量PCR（RT—PCR）测定血管内皮生长因子-A（VEGF—A）、血管生成素-2（Ang-2）、基质金属蛋白酶-2（MMP-2）、MMP-9mRNA表达。C57BL／6小鼠接种LLC后随机分为3组,每组6只,分别予以瘤内注射PBS、对照腺病毒（Ad—NC）和干扰腺病毒（Ad—si—IL-17a,IL-17a基因沉默）,16d后处死小鼠,提取肿瘤组织RNA,RT-PCR检测VEGF-A、Ang-2、MMP-2和MMP-9mRNA表达。结果体外培养条件下IL-17A对肿瘤细胞的增殖能力没有影响,与无IL-17A组相比,50μg／L浓度的IL-17A组的肿瘤细胞VEGF-A、Ang-2、MMP-2和MMP-9mRNA表达水平明显升高,且肿瘤细胞侵袭能力增强（P〈0．01）。Ad—si—IL-17a组小鼠在13d（P〈0．05）和16d（P〈0．01）时肿瘤明显小于Ad-NC组和PBS组。Ad-si-IL-17a组肿瘤VEGF-A、Ang-2、MMP-2和MMP-9ITIRNA表达水平低于Ad—NC组和PBS组。结论靶向IL-17A的治疗可能为肿瘤治疗提供新思路。     

Lipid hydroperoxide stimulates retinal neovascularization in rabbit retina through expression of tumor necrosis factor-a, vascular endothelial growth factor and platelet-derived growth factor

To test the hypothesis that oxidative damage associated with tissue hypoxia plays a role in neovascularization, a lipid hydroperoxide (LHP) was injected into the vitreous of rabbits. Single injections of LHP (50–600g) caused a sustained retinal neovascularization visualized clinically by ophthalmoscopy and confirmed by microscopy. Vasodilators, i.e. histamine and nitric oxide, peaked at 6h and 7 days, respectively. The levels of both tumor necrosis factor- and interleukin-l peaked at 12h and dropped to basal levels by 24h. Expression of vascular endothelial growth factor (VEGF) and transforming growth factor- peaked at 24h and were sustained throughout the following 3 weeks, and platelet-derived growth factor was also elevated throughout the same period. Upregulation of these five angiogenic cytokines, but not basic fibroblast growth factor, occurred prior to the appearance of neovascularization. Leakage of fluorescein at the tips of new vessels was demonstrated by fluorescein angiography. Linoleic hydroperoxide induced neovascularization, but saturated or unsaturated native C-18 fatty acids had no effect. The cascade of multiple, angiogenic cytokines induced by LHP may interact to promote sustained neovascularization.     

A permissive role for tumor necrosis factor in vascular endothelial growth factor-induced vascular permeability

Vascular endothelial growth factor (VEGF) induces both angiogenesis and an increase in vascular permeability, 2 processes that are considered to be important for both tumor growth and the delivery of drugs to the site of tumors. This study demonstrates that transmembrane expression of tumor necrosis factor (tmTNF) is up-regulated in the endothelium of a murine methylcholanthrene (meth A)-induced sarcoma in comparison to the adjacent normal dermal vasculature and is also present on cultivated human endothelial cells. It is further shown that tmTNF is required for VEGF-mediated endothelial hyperpermeability in vitro and in vivo. This permissive activity of TNF appears to be selective, because anti-TNF antibodies ablated the VEGF-induced permeability but not proliferation of cultivated human endothelial cells. Furthermore, tnf gene-deficient mice show no obvious defects in vascularization and develop normally but failed to respond to administration of VEGF with an increase in vascular permeability. Subsequent studies indicated that the tmTNF and VEGF signaling pathways converge at the level of a secondary messenger, the "stress-activated protein kinase-2" (SAPK-2)/p38: (1) up-regulated endothelial expression of tmTNF resulted in the continuous activation of SAPK-2/p38 in vitro, and (2) an inhibitor of SAPK-2/p38 activation abolished the vascular permeability activity of VEGF in vivo. In conclusion, the study's finding that continuous autocrine signaling by tmTNF sensitizes endothelial cells to respond to VEGF by increasing their vascular permeability provides new therapeutic concepts for manipulating vascular hyperpermeability.     

VEGF对血管内皮细胞损伤的拮抗作用及作用机制

冻结性冷损伤是以组织冻结再融化过程的病理改变为基础的损伤。已经证实,血管内皮细胞（VECs）除有屏障和膜转运功能外,还具有内分泌功能,主动参与了多种生命活动,对维持体内环境的平衡与稳定具有重要作用。冻伤过程中VECs的损伤,可促进微循环血栓形成造成微循环障碍;待到复温后,血液恢复流动又可导致不可逆的再灌注损伤。因此,减轻内皮细胞损伤,可以减少微循环血栓形成,从而减轻再灌注对机体造成的损伤。血管内皮细胞生长因子（VEGF）作为重要的血管生长因子,在冻伤时对VECs损伤具有拮抗作用,可通过促进血管再生、抗血栓形成、抑制血管平滑肌过度生长及抗炎的作用等实现的对血管的保护作用,为临床防治冻伤提供了新的思路。     

Interplay between EphB4 on tumor cells and vascular ephrin-B2 regulates tumor growth

Receptor tyrosine kinases of the Eph family are up-regulated in different types of cancer. EphB4 and its ligand ephrin-B2 have been linked to breast cancer, but little is known about how this receptor-ligand complex may contribute to oncogenesis. The Eph receptors transmit forward signals via their kinase domain and reverse signals via their transmembrane ephrin-B ligands. Therefore, we used EphB4 that were lacking the kinase domain and tagged with EGFP (EphB4 Delta C-EGFP) to differentiate between EphB4 and ephrin-B2 signaling. Interestingly, we found that expression of EphB4 Delta C-EGFP in breast cancer cells increases tumor growth in a mouse xenograft model. Given the undetectable EphB4 activation in the tumor cells, dominant negative effects of EphB4 Delta C-EGFP are unlikely to explain the increased tumor growth. Examination of the tumors revealed that ephrin-B2 is primarily expressed in the vasculature and that the EphB4 Delta C-EGFP tumors have a higher blood content than control tumors, concomitant with increased size of blood vessels. In support of an effect on the vasculature, the extracellular domain of EphB4 attracts endothelial cells in vitro and stimulates endothelial cell invasion, survival, and proliferation, all crucial factors for angiogenesis. These results support a model in which EphB4 promotes tumor growth by stimulating angiogenesis through ephrin-B2.     

Defense mechanism and macroscopic tumor growth in lung tissue

A total of 126 resection specimens from malignant lung tumors were cut into serial sections, and tumor volume and macroscopic growth pattern were computed. Four characteristic tumor growth patterns could be separated: Tumors growing in bizarre, irregular shapes; Tumors growing in spheroid shapes; Tumors growing in ellipsoid shapes; Tumors growing in mixed growth pattern. The immunologic response of the host tissue was analyzed grading the number of lymphocytes, plasma cells, macrophages in and at the boundary of the tumor tissue. Lymphocytic subpopulations were analyzed in 46 cases using monoclonal antibodies (BS3/BS4; T3, OKT4, OKT8, OKT11, OKT14). The majority of lymphocytes were T-lymphocytes and monocytes in cases with inflammatory response of host tissue. The ratio of inducer/helper subset (OKT4+) compared to suppressor/cytotoxic subset (OKT8+) was similar in expression as reported for circulating peripheral T-lymphocytes. The different growth patterns depend upon cell type of tumor, immunologic response of the host tissue, and tumor volume. The findings indicate that tumor progression into lung tissue is partly due to "localized metastatic growth" of different tumor cell subpopulations.     

Nine-year patency of a vascular prosthesis used for portal vein reconstruction during pancreatoduodenectomy

We report a patient in whom a polytetrafluoroethylene (PTFE) graft used for reconstruction of the portal vein was confirmed to be patent 9 years after pancreatoduodenectomy (which was performed when he was aged 51 years). The patient had advanced cancer of the head of the pancreas. Pancreatoduodenectomy was performed, and 6 cm of the portal vein was resected. The portal vein was reconstructed with a PTFE graft (internal diameter 9 mm; length about 6 cm). The graft was demonstrated to be patent on angiography 3 years after the operation. A computed tomographic (CT) scan performed 9 years after the operation showed that the portal graft was still patent. About 2 years after the operation, the patient had been able to resume physical labor, similar to the work he performed before the operation.     

The effect of antibody against vascular endothelial growth factor on tumor growth and metastasis

Vascular endothelial growth factor (VEGF), a very important in the process of tumor angiogenesis, was chosen as a target in a study to determine whether manipulation of angiogenesis with antibody against VEGF may interrupt tumor growth and metastasis. Anti-VEGF antibody was obtained from immunized rabbits, purified on an affinity column, and identified as neutralized antibody by Mile's assay. IVTA₂MA891, a murine spontaneous breast cancer with a high rate of metastasis in lung in TA₂ × 615 F1 mice, was chosen as an animal model in this study, because of the high expression of VEGF in the primary tumor as well as in the lung metastatic tumor. The anti-VEGF antibody could inhibit growth of S180 sarcoma in a dose-dependent manner, and the inhibition rate could reach 41.0% with a dose of 200 μg mouse⁻¹ day⁻¹. Anti-VEGF antibody could inhibit tumor growth by 76.2% in nude mice bearing human gastric cancer (MGC 803). When anti-VEGF antibody was combined with ¹³¹I-3H11, a murine monoclonal antibody conjugated with ¹³¹I, only one of five nude mice developed tumor and 84.0% more inhibition of tumor growth was obtained in comparison with treatment by ¹³¹I-3H11 alone. The growth of the primary tumor was inhibited by 44.0% and the number and size of the metastatic foci in the lungs were reduced by 73.0% and 83.7% respectively in the animal model, with a high rate of metastasis in lung. The anti-VEGF antibody may be potentially useful for clinical treatment of cancer and metastasis. Received: 2 January 1998 / Accepted: 17 March 1998     

Peptide vaccination can lead to enhanced tumor growth through specific T-cell tolerance induction.

Vaccination with synthetic peptides representing cytotoxic T lymphocyte (CTL) epitopes can lead to a protective CTL-mediated immunity against tumors or viruses. We now report that vaccination with a CTL epitope derived from the human adenovirus type 5 E1A-region (Ad5E1A234-243), which can serve as a target for tumor-eradicating CTL, enhances rather than inhibits the growth of Ad5E1A-expressing tumors. This adverse effect of peptide vaccination was rapidly evoked, required low doses of peptide (10 micrograms), and was achieved by a mode of peptide delivery that induces protective T-cell-mediated immunity in other models. Ad5E1A-specific CTL activity could no longer be isolated from mice after injection of Ad5E1A-peptide, indicating that tolerization of Ad5E1A-specific CTL activity causes the enhanced tumor outgrowth. In contrast to peptide vaccination, immunization with adenovirus, expressing Ad5E1A, induced Ad5E1A-specific immunity and prevented the outgrowth of Ad5E1A-expressing tumors. These results show that immunization with synthetic peptides can lead to the elimination of anti-tumor CTL responses. These findings are important for the design of safe peptide-based vaccines against tumors, allogeneic organ transplants, and T-cell-mediated autoimmune diseases.