Expression of tissue factor in pancreatic adenocarcinoma is associated with activation of coagulation

AIM: To study expression of tissue factor (TF) in pancreatic cancer and its role in the development of thromboembolism. METHODS: TF expression was studied in eight human pancreatic carcinoma cell lines by Northern blot and indirect immunofluorescence. Expression of alternatively spliced TF (asTF) was assessed by RTPCR. In addition, TF expression was determined by immunofluorescence in pancreatic tissues of 19 patients with pancreatic adenocarcinoma (Pca), 9 patients with chronic pancreatitis (CP) and 20 normal controls. Plasma samples (30 Pca-patients, 13 CP-patients and 20 controls) were investigated for soluble TF levels and coagulation activation markers [thrombin-antithrombin Ⅲ complex (TAT), prothrombin fragment 1 + 2 (F1 + 2)]. RESULTS: All pancreatic carcinoma cell lines expressed TF (8/8) and most of them expressed asTF (6/8). TF expression at the protein level did not correlate with the differentiation of the carcinoma cell line. All but two pancreatic cancer tissue samples stained positive for TF (17/19). In all samples of CP weak staining was restricted to pancreatic duct cells, whereas only a few subendothelial cells were positive in 9/20 of normal controls. TF and TAT levels in Pca patients were significantly elevated compared to controls whereas elevated F1 + 2 levels did not reach statistical significance compared to controls. In CP patients TAT and F1 + 2 levels proved to be significantly elevated compared to controls, although TAT elevation was less pronounced than in Pca patients. CONCLUSION: We conclude that in addition to the upregulated expression of TF on the cell membrane, soluble TF might contribute to activation of the coagulation system in pancreatic cancer.     

Cocaine unbalances endothelial tissue factor and tissue factor pathway inhibitor expression

Cocaine consumption can lead to myocardial infarction. Tissue factor (TF) has been implicated in acute coronary syndromes, and the balance of TF and tissue factor pathway inhibitor (TFPI) determines initiation of thrombus formation. This study was designed to investigate the effect of cocaine on endothelial TF and TFPI expression. Cocaine (10(-8)-10(-5) mol/l) increased thrombin-induced TF expression by 24% at 10(-7) mol/l (P < 0.001) without affecting basal TF expression. In contrast, cocaine reduced endothelial TFPI expression by 47% at 10(-7) mol/l (P < 0.01). Moreover, thrombin impaired endothelial TFPI expression, and cocaine (10(-8) mol/l) further reduced TFPI expression by 33% as compared to thrombin (P < 0.02). These effects occur at cocaine concentrations usually present in plasma of consumers. Given the importance of TF in the pathogenesis of acute coronary syndromes, TF induction in conjunction with TFPI suppression may be relevant for the increased frequency of myocardial infarction observed in cocaine consumers.     

Expression of fibroblast activation protein in human pancreatic adenocarcinoma and its clinicopathological significance

AIM: To examine fibroblast activation protein (FAP) expression in pancreatic ductal adenocarcinoma (PDAC) and to analyze its relationship with the clinicopathology of PDAC.METHODS: FAP expression was examined in 134 PDAC specimens by immunohistochemistry, and in four pancreatic cancer cell lines (SW1990, Miapaca-2, AsPC-1 and BxPC-3) by Western blotting assay. We also analyzed the association between FAP expression in PDAC cells and the clinicopathology of PDAC patients.RESULTS: The results showed that the FAP was ex-pressed in both stromal fibroblast cells (98/134, 73.1%) and carcinoma cells (102/134, 76.1%). All 4 pancreatic cancer cell lines expressed FAP protein at different levels. Protein bands corresponding to the proteolytically active 170-kDa seprase dimer and its 88-kDa seprase subunit were identified. Higher FAP expression in carcinoma cells was associated with tumor size (P < 0.001), fibrotic focus (P = 0.003), perineural invasion (P = 0.009) and worse clinical outcome (P = 0.0085).CONCLUSION: FAP is highly expressed in carcinoma cells and fibroblasts in PDAC tissues, and its expression is associated with desmoplasia and worse prognosis.     

Diabetes associated with pancreatic ductal adenocarcinoma is just diabetes: Results of a prospective observational study in surgical patients

BackgroundIdentification of a specific diabetes signature associated to pancreatic ductal carcinoma (PDAC) could be a key to detect asymptomatic, early stage tumors. We aim to characterize the clinical signature and the pathogenetic factors of the different types of diabetes associated with PDAC, based on the time between diabetes and cancer diagnosis.MethodsProspective observational study on 364 PDAC patients admitted to a referral center for pancreatic disease. Hospital and/or outpatient medical records were reviewed. Blood biochemical values including fasting blood glucose, insulin and/or C-peptide, glycosylated hemoglobin and anti-islet antibodies were determined. Diabetes onset was assessed after surgery and during follow-up.ResultsThe prevalence of diabetes in patients was 67%. Considering 174 patients (47.8%) already having diabetes when diagnosed with PDAC (long duration, short duration, concomitant), the clinical and biochemical profile was similar to that of patients with type 2 diabetes (T2D). Diabetes was associated with known risk factors (i.e., age, sex, family history for diabetes and increased BMI) and both beta-cell dysfunction and insulin resistance were present. Considering 70 patients (19.2%) with onset of diabetes after PDAC diagnosis (early and late onset), the strongest predictor was the loss of beta-cell mass following pancreatectomy in patients with risk factors for T2D.ConclusionDifferent types of diabetes according to the time between diabetes and PDAC diagnosis are clinical entities widely overlapping with T2D. Therefore, the success of a strategy considering diabetes onset as a marker of asymptomatic PDAC will largely depend on our ability to identify new diabetes-unrelated biomarkers of PDAC.     

Notch pathway activation is associated with pancreatic cancer treatment failure

BackgroundPancreatic cancer is resistant to conventional treatment. The aim of the study was to confirm the hypothesis that changes in cancer stem cells (CSCs) and developmental pathway after treatment was responsible for treatment failure in pancreatic cancer.MethodsAfter recovery from a gemcitabine treatment, the percentage of pancreatic cancer CSCs and Notch pathway in BxPC3 and HPAC pancreatic cancer cell lines were analyzed by FACS (CD24 and CD44) and western blot (Notch1, Hes1, β-catenin, and pAKT). The effect of DAPT, a gamma-secretase inhibitor, was similarly investigated. The association between immunohistochemical expression of Hes1 and survival was analyzed.ResultsThe percentage of CD24⁺CD44⁺ cells was higher in gemcitabine-treated BxPC3 and HPAC cells than at pre-treatment. CD24⁺CD44⁺ cells sorted from the gemcitabine-treated cell lines showed higher migration and invasion ability than CD24⁻CD44⁻ or CD24⁻CD44⁺ cells from the same cell lines. Western blot analysis showed an increased expression of Notch1 and Hes1 in gemcitabine-treated cell lines. The overall survival of pancreatic cancer patients with strong expression of Hes1 was shorter than that in patients with no or weak expression (11.1 vs. 21.6 months, P = 0.036). Treatment with DAPT reversed the increase in Hes1, β-catenin, and pAKT expression and the proportion of CD24⁺CD44⁺ cells in gemcitabine-treated cell lines. The treatment also decreased migration and invasion ability.ConclusionOur data suggested that an increase in CSCs and activation of the Notch pathway might contribute to the failure of treatment in pancreatic cancer. Notch pathway can be a potential target to overcome treatment failure.     

Increased platelet,leukocyte, and coagulation activation in primary myelofibrosis

Platelet and leukocyte activation has been demonstrated in polycythemia vera (PV) and essential thrombocythemia (ET), but such information is limited in primary myelofibrosis (PMF). Platelet, leukocyte, endothelial, and coagulation activation status was assessed in 26 PMF patients and compared with data from 22 age- and sex-matched healthy individuals. Study included flow cytometry assessment of platelet P-selectin expression [at baseline and after adenosine diphosphate (ADP), thrombin and arachidonic acid stimulation], platelet–neutrophil and platelet–monocyte complexes, and CD11b expression in neutrophils and monocytes. Additionally, soluble P-selectin, sCD40L, tissue factor, thrombomodulin, prothrombin fragment 1 + 2 (F1 + 2), and D-dimer were measured by enzyme-linked immunosorbent assays. The above parameters were correlated with the patients’ clinical data and presence of the JAK2 V617F mutation. Compared with controls, PMF patients had increased baseline platelet activation, as shown by significantly higher levels of soluble and platelet P-selectin expression, and also higher percentages of platelet–monocyte complexes. Neutrophil and monocyte CD11b expression was significantly higher in patients with the JAK2 mutation than in those with wild-type allele or the controls. Endothelial and coagulation activation, as demonstrated by increased plasma levels of thrombomodulin and F1 + 2, was also found in PMF, with patients with the JAK2 mutation showing significantly higher values of F1 + 2 than those with wild-type allele. In conclusion, PMF patients have platelet, leukocyte, endothelial, and coagulation activation similar to that in PV and ET. CD11b overexpression and F1 + 2 are correlated with the presence of the JAK2 mutation.     

冠心病患者血浆组织因子和组织因子途径抑制物的变化

目的 :通过检测不同类型冠心病 (CHD)患者血浆组织因子 (TF)和组织因子途径抑制物 (TFPI)水平变化 ,探讨其在CHD发病过程中的作用。方法 :以酶联免疫吸附测定法测定CHD患者血浆中TF和TFPI抗原水平。结果 :不稳定型心绞痛 (UAP)和急性心肌梗死 (AMI)患者的血浆TF和TFPI水平与正常对照者和稳定型心绞痛 (SAP)患者相比均有显著性增高 (P <0 .0 5 ) ,以AMI患者尤为明显 (P <0 .0 1) ;UAP和AMI患者的TF PI/TF比值显著降低 (P <0 .0 5 ) ,而SAP患者的上述指标与正常对照者相比 ,其差异均无显著性意义 (P >0 .0 5 )。结论 :UAP和AMI患者TFPI/TF系统失衡 ,标志高凝状态的存在 ;TF和TFPI在这两种类型CHD的发病机制中可能起着重要的作用     

胰腺纤维化后结缔组织生长因子的表达及意义

目的 观察胰腺纤维化后结缔组织生长因子(connective tissue growth factor,CTGF)在胰腺组织内的表达,探讨其意义。方法 通过高脂饲料诱导大鼠胰腺纤维化模型,16周后处死大鼠,取胰腺组织常规病理检查,天狼猩红染色和免疫组织化学染色检测胰腺纤维化组织胶原蛋白I、α-SMA及CTGF蛋白表达。结果 胰腺纤维化后,胰腺小叶和腺泡萎缩,小叶间隙增宽,间质内纤维组织明显增生;胰腺组织内胶原蛋白Ⅰ的合成较正常胰腺明显增加(1207.3±115.5比166.7±78.4,P＜0.01),α-SMA 表达量较正常胰腺组织增高(1500.2±255.8比57.4±23.2,P＜0.01),CTGF表达较正常胰腺明显增加(2950.5±431.9比382.2±190.8,P＜0.01),且胰腺星状细胞(PSCs)大量活化。结论 CTGF是胰腺纤维化的重要作用因子,其作用与PSCs活化密切相关。     

ApoE-/-小鼠动脉粥样硬化病变中稳定斑块组织因子的表达

目的:研究稳定斑块组织因子(TF)活化表达及在血栓形成中的作用。方法:建立ApoE-/-小鼠动脉粥样硬化病变的动物模型,一期凝固法检测小鼠血浆TF特异性的促凝活性,光镜和电镜观察稳定斑块病变,免疫组化SP法检测小鼠稳定斑块TF的蛋白表达,原位杂交法检测小鼠稳定斑块TF mRNA表达。结果:ApoE-/-小鼠血浆促凝活性显著增加(P<0.01),并随着诱发斑块时间的延长呈上升趋势,抗TF单抗能显著抑制血浆促凝活性(P<0.01);实验小鼠的病变为稳定斑块,电镜下,ApoE-/-小鼠可见完整的血管内皮细胞,在内皮完整的血管内有血小板的聚集;不同狭窄程度的ApoE-/-小鼠动脉粥样硬化病变部位TF的表达差异有统计学意义(P<0.01),TF的表达随着粥样硬化病变的进展而增强,TFmRNA与TF蛋白表达一致。结论:TF可能通过活化和释放启动凝血过程,导致血栓的形成;TF不仅是粥样硬化导致动脉损伤的结果,还可能是促进动脉粥样硬化病变的重要原因。     

Clinical significance of expression of tissue factor and tissue factor pathway inhibitor in ulcerative colitis

AIM: To investigate the clinical significance of expression of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) in ulcerative colitis (UC).METHODS: Thirty UC specimens taken by colonoscopy from patients with active UC treated at the Department of Pathology, Central Hospital Affiliated to Shenyang Medical College from February 2010 to January 2012 were included in an experimental group, and 30 normal colon tissue samples taken by colonoscopy from non-UC patients were included in a control group. Expression of TF and TFPI in UC and normal colon tissue samples was detected by immunohistochemistry.RESULTS: The positive rate of TF in UC was significantly higher than that in normal colon tissue (63% vs 33%, χ² = 5.41, P < 0.05). The positive rate of TFPI in UC was also significantly higher than that in normal colon tissue (43% vs 17%, χ² = 5.08, P < 0.05).CONCLUSION: Positive rates of TF and TFPI expression in UC are significantly higher than those in normal colon tissue. TF and TFPI may play an important role in the pathogenesis of UC.     

In vivo coagulation activation following infusion of highly purified factor XI concentrate

The use of factor XI concentrates has been associated with thrombosis. Plasma markers of coagulation activation were measured before and 30, 60, 120 and 240 min after six infusions of the BPL factor XI concentrate. Five studies were completed before surgical intervention, one was undertaken in a patient with an intracerebral haemorrhage. Significant elevation of levels of fibrinopeptide A (FpA) ( P  < 0.05) and thrombin–antithrombin (TAT) were demonstrated following six infusions and prothrombin fragment F1.2 following four. Levels of all three markers had risen 60 min following concentrate administration and FpA levels remained elevated throughout the study period. Levels of D-dimer rose in four patients at 240 min. These results indicate significant thrombin generation by 60 min and subsequent plasmin generation consistent with coagulation activation by the factor XI concentrate. The greatest elevation of activation markers was seen in those subjects with pre-existing coagulation activation. We advise caution in the use of these products and awareness of the risks in patients who may already have activated coagulation states.     

胰腺癌组织表皮生长因子mRNA表达的意义

目的探讨ＥＧＦｍＲＮＡ的表达与胰腺癌发生、发展及预后的关系．方法应用Ｎｏｒｔｈｅｒｎｂｌｏｔ杂交方法，检测胰腺癌２７例和正常胰组织７例中ＥＧＦｍＲＮＡ表达．结果正常胰腺组织７例未检测到ＥＧＦｍＲＮＡ表达，胰腺癌２７例ＥＧＦｍＲＮＡ阳性表达率６６７％（１８例），经卡方检验发现，ＥＧＦｍＲＮＡ表达与胰腺癌病理分级、临床分期及伴随局部淋巴结转移呈显著相关（Ｐ＜００５）．结论ＥＧＦｍＲＮＡ表达与胰腺癌发生有关，胰腺癌ＥＧＦｍＲＮＡ表达可作为胰腺癌预后的参考指标     

凝血因子VR2等位基因多态性在血栓相关性疾病患者中的分布

目的：通过检测血栓相关性疾病患者的凝血因子V（coagulation factorV)R2等位基因多态性，探讨R2单体型与血栓形成的相关性。方法：采用PCR-酶切法对100个脑血栓患者，96个心肌梗塞患者，45个深静脉血栓患者，80个系统性红斑狼疮患者，以及98个正常对照进行FVR2等位基因检测。结果：首次发现2例系统性红斑狼疮患者基因型为R2等位基因杂合子。结论：中国汉族人群血栓形成的遗传分子背景与FVHR2等位基因可能没有相关性。     

维生素E对组织因子及其抑制物在冠心病中的干预作用

目的 :探讨冠心病 (CHD)的不同类型中血浆组织因子 (TF)及其抑制物 (TFPI)含量的差异变化及维生素E对急性心肌梗死 (AMI)干预作用。方法 :用ELISA方法检测CHD患者 [包括稳定型心绞痛 (SAP)、不稳定型心绞痛 (UAP)、AMI]入院时及治疗 2周时血浆TF、TFPI含量。结果 :①SAP组血浆TF、TFPI水平及TF/TF PI比值均高于正常对照组 ,但差异无统计学意义 (P >0 .0 5 ) ,且治疗前后无明显变化 (P >0 .0 5 )。②UAP组、AMI组血浆TF、TFPI水平及TF/TFPI比值明显高于正常对照组 ,差异有统计学意义 (P <0 .0 1) ,但两组常规治疗前后差异无统计学意义 (P >0 .0 5 )。③AMI加维生素E组干预治疗后TF值显著下降 (P <0 .0 1) ,与正常对照组比较差异无统计学意义 (P >0 .0 5 ) ,TFPI干预治疗后无明显变化 (P >0 .0 5 ) ,而TF/TFPI比值明显降低 (P <0 .0 1)。④各组TF与TFPI呈明显正相关 (P<0 .0 5 ,r=0 .4 32 )。结论 :TF及TFPI在CHD尤其是急性冠状动脉综合征的发生中起重要作用 ,维生素E的干预显著降低患者血浆TF水平 ,降低TF/TFPI比值而对TFPI无影响。     

神经生长因子及其受体在人胰腺导管癌组织中的表达

目的 探讨β神经生长因子(β-NGF)及其受体[酪氨酸激酶A(tyrosine kinase A,TrKA)和P75NGFR]在人胰腺导管癌组织中的表达及意义.方法 选择胰腺导管癌组织标本80例和正常胰腺组织20例,采用免疫组化、荧光定量PCR等方法,定量检测β-NGF及其受体表达,分析β-NGF)及其受体与临床病理学特征,尤其与神经侵袭的关系.结果 随着癌细胞分化程度降低和肿瘤TNM分期增加,β-NGF、TrKA蛋白表达水平增加,且低分化与高分化、中分化组相比显著增加(P<0.01),Ⅲ～Ⅳ期较Ⅰ～Ⅱ期显著增加(P<0.05);β-NGF、TrKA蛋白表达水平在有神经浸润组明显高于无神经浸润组(P<0.01),有淋巴结转移组明显高于无淋巴结转移组(P<0.01),且邻近神经组织的肿瘤细胞β-NGF、TrKA阳性程度高于远离神经组织的癌细胞.P75NGFR表达与肿瘤细胞分化程度关系密切.β-NGF和TrKA表达呈正相关.β-NGF、TrKA和P75NGFR mRNA表达水平在胰腺癌组织中明显高于正常胰腺组织,分别增加3.84、4.23和2.41倍.结论 β-NGF及其受体参与介导胰腺癌的发生,与胰腺癌临床病理学特征关系密切,β-NGF、TrKA高表达和胰腺癌嗜神经性关系密切.     

Low levels of activated factor VII in systemic sclerosis

Introduction. Recent investigations show that activated factor VII, the primary enzyme in the extrinsic pathway of blood coagulation, exerts additional extra-coagulant functions, such as apoptosis and angiogenesis. On the basis of these recent acquisitions, the present study was aimed to evaluate activated factor VII in patients with systemic sclerosis and to establish a potential association with pathogenesis and complications of this severe autoimmune disorder. Materials and methods. Activated factor VII level was measured in twenty-eight consecutive scleroderma patients (2 men and 26 women, mean age 49.7 ± 14.8 years). The main clinical correlates of disease, such as disease activity, renal function, skin, vascular and lung involvement, were evaluated by clinical and instrumental investigations. Activated factor VII level was also evaluated in 28 sex and age matched controls. Results. Systemic sclerosis patients exhibited plasma activated factor VII activities significantly lower than those of healthy matched controls (15.2 versus 37.7 U/l, respectively; p < 0.001). No correlation was observed between plasma activated factor VII concentration and age, disease duration, disease subset, disease activity, renal, lung, skin and microvascular involvement. Conclusions. Results of our investigation provide first evidence of low activated factor VII activity in patients with systemic sclerosis. Reduced activated factor VII activity might be involved in the pathogenesis of the ischemic complications, by modulating apoptotic and angiogenetic processes. Contributed equally to this work.     

Tumor budding is a prognostic factor linked to epithelial mesenchymal transition in pancreatic ductal adenocarcinoma. Study report and literature review

Background

Pancreatic ductal adenocarcinoma (PDAC) has a devastatingly poor prognosis. Surgical resection is undertaken in only 20% of patients. Most of well-known prognostic factors reflect tumor stage more than its biology. So it is important to identify new biological indicators related to survival in order to develop new therapies.

胰腺癌组织中人粒细胞集落刺激因子-1的表达及意义

目的 研究人粒细胞集落刺激因子（hGC）-1在胰腺癌组织中的表达及其与胰腺癌临床病理参数的相关性,探讨hGC-1的表达与胰腺癌发生发展的相关性.方法 采用免疫组化SP法检测31例人胰腺导管细胞癌及其对应癌旁组织标本中hGC-1的表达,并结合临床病理资料进行相关性分析.结果 胰腺癌组织中hGC-1的表达水平明显高于癌旁组织,且表达强度与TNM分期和有无淋巴结转移密切相关（P ＜0.05）;31例胰腺癌组织中hGC-1的表达水平与患者性别、年龄无明显相关（P＞0.05）.结论 hGC-1表达上调与胰腺癌的发生、发展密切相关,hGC-1可作为反映胰腺癌恶性生物学行为的1个指标.     

Preclinical evaluation of herpes simplex virus armed with granulocyte-macrophage colony-stimulating factor in pancreatic carcinoma

AIM: To investigate the therapeutic efficacy and mechanisms of action of oncolytic-herpes-simplex-virus encoding granulocyte-macrophage colony-stimulating factor(HSVGM-CSF) in pancreatic carcinoma.METHODS: Tumor blocks were homogenized in a sterile grinder in saline.The homogenate was injected into the right armpit of each mouse.After vaccination,the mice were randomly assigned into four groups: a control group,a high dose HSVGM-CSFgroup [1 × 107plaque forming units(pfu)/tumor],a medium dose HSVGM-CSF group(5 × 106pfu/tumor) and a low dose HSVGM-CSF group(5 × 105pfu/tumor).After initiation of drug administration,body weights and tumor diameters were measured every 3 d.Fifteen days later,after decapitation of the animal by cervical dislocation,each tumor was isolated,weighed and stored in 10% formaldehyde solution.The drug effectiveness was evaluated according to the weight,volume and relative volume change of each tumor.Furthermore,GM-CSF protein levels in serum were assayed by enzyme-linked immunosorbent assays at 1,2,3 and 4 d after injection of HSVGM-CSF.RESULTS: Injection of the recombinant mouse HSV encoding GM-CSF resulted in a significant reduction in tumor growth compared to the control group,and dosedependent effects were observed: the relative tumor proliferation rates of the low dose,medium dose and high dose groups on 15 d after injection were 45.5%,55.2% and 65.5%,respectively.The inhibition rates of the tumor weights of the low,middle,and high dose groups were 41.4%,46.7% and 50.5%,respectively.Furthermore,the production of GM-CSF was significantly increased in the mice infected with HSVGM-CSF.The increase in the GM-CSF level was more pronounced in the high dose group compared to the other two dose groups.CONCLUSION: Our study provides the first evidence that HSVGM-CSFcould inhibit the growth of pancreatic cancer.The enhanced GM-CSF expression might be responsible for the phenomenon.