Changes in Vascular Permeability and Expression of Different Angiogenic Factors Following Anti-Angiogenic Treatment in Rat Glioma

Background

Anti-angiogenic treatments of malignant tumors targeting vascular endothelial growth factor receptors (VEGFR) tyrosine kinase are being used in different early stages of clinical trials. Very recently, VEGFR tyrosine kinase inhibitor (Vetanalib, PTK787) was used in glioma patient in conjunction with chemotherapy and radiotherapy. However, changes in the tumor size, tumor vascular permeability, vascular density, expression of VEGFR2 and other angiogenic factors in response to PTK787 are not well documented. This study was to determine the changes in tumor size, vascular permeability, fractional plasma volume and expression of VEGFR2 in PTK787 treated U-251 glioma rat model by in vivo magnetic resonance imaging (MRI) and single photon emission computed tomography (SPECT). The findings were validated with histochemical and western blot studies.

Methodologies and Principal Findings

Seven days after implantation of U251 glioma cells, animals were treated with either PTK787 or vehicle-only for two weeks, and then tumor size, tumor vascular permeability transfer constant (K^trans), fractional plasma volume (fPV) and expression of VEGFR2 and other relevant angiogenic factors were assessed by in vivo MRI and SPECT (Tc-99-HYNIC-VEGF), and by immunohistochemistry and western blot analysis. Dynamic contrast-enhanced MRI (DCE-MRI) using a high molecular weight contrast agent albumin-(GdDTPA) showed significantly increased K^trans at the rim of the treated tumors compared to that of the central part of the treated as well as the untreated (vehicle treated) tumors. Size of the tumors was also increased in the treated group. Expression of VEGFR2 detected by Tc-99m-HYNIC-VEGF SPECT also showed significantly increased activity in the treated tumors. In PTK787-treated tumors, histological staining revealed increase in microvessel density in the close proximity to the tumor border. Western blot analysis indicated increased expression of VEGF, SDF-1, HIF-1α, VEGFR2, VEGFR3 and EGFR at the peripheral part of the treated tumors compared to that of central part of the treated tumors. Similar expression patters were not observed in vehicle treated tumors.

Conclusion

These findings indicate that PTK787 treatment induced over expression of VEGF as well as the Flk-1/VEGFR2 receptor tyrosine kinase, especially at the rim of the tumor, as proven by DCE-MRI, SPECT imaging, immunohistochemistry and western blot.     

Interpretation of Unexpectedly High Levels of Endocrine Tumor Markers

Objective: Endocrine tumor markers (ETMs) are important and indispensable tools in the diagnosis and follow-up of endocrine tumors. Unexpectedly high level of ETM (UHLETM) is often encountered in clinical practice. The objectives of this article are to discuss the approach to UHLETMs and describe the most common UHLETMs.Methods: Literature review and personal experience with UHLETMs.Results: A UHLETM is defined as an ETM level much higher than what an endocrinologist would expect based on the patient's clinical information, other biochemical test results, and imaging findings. Most UHLETMs are false positive, in that they do not indicate the existence or growth of an endocrine tumor. The key issue, however, is how to convincingly prove that a UHLETM is false positive. The most important question to help resolve UHLETMs is whether the UHLETMs are due to false or true results. Some UHLETMs are due to interpretation errors, laboratory errors, or spurious test results, while the true levels of the ETMs are normal or unchanged from previous results. Other UHLETMs are due to nontumor conditions or medications, and the true levels of the ETMs are indeed very high. When it is not straightforward to assess whether certain UHLETMs are due to false or true results, they may be due to novel conditions.Conclusion: UHLETMs provide endocrinologists great opportunities to learn the basic biology and measurement of ETMs. Unresolved UHLETMs are exciting clinical research opportunities which may lead to discovery of new diseases and new mechanisms of measurement interference.Abbreviations: 5-HIAAA = 5-hydroxyindoleacetic acid; 5-HTP = 5-hydroxytryptophan; CGA = chromogranin A; ETM = endocrine tumor marker; MTC = medullary thyroid carcinoma; UHLETM = unexpectedly high level of endocrine tumor marker     

Differential DNA Methylation Correlates with Differential Expression of Angiogenic Factors in Human Heart Failure

Epigenetic mechanisms such as microRNA and histone modification are crucially responsible for dysregulated gene expression in heart failure. In contrast, the role of DNA methylation, another well-characterized epigenetic mark, is unknown. In order to examine whether human cardiomyopathy of different etiologies are connected by a unifying pattern of DNA methylation pattern, we undertook profiling with ischaemic and idiopathic end-stage cardiomyopathic left ventricular (LV) explants from patients who had undergone cardiac transplantation compared to normal control. We performed a preliminary analysis using methylated-DNA immunoprecipitation-chip (MeDIP-chip), validated differential methylation loci by bisulfite-(BS) PCR and high throughput sequencing, and identified 3 angiogenesis-related genetic loci that were differentially methylated. Using quantitative RT-PCR, we found that the expression of these genes differed significantly between CM hearts and normal control (p<0.01). Moreover, for each individual LV tissue, differential methylation showed a predicted correlation to differential expression of the corresponding gene. Thus, differential DNA methylation exists in human cardiomyopathy. In this series of heterogenous cardiomyopathic LV explants, differential DNA methylation was found in at least 3 angiogenesis-related genes. While in other systems, changes in DNA methylation at specific genomic loci usually precede changes in the expression of corresponding genes, our current findings in cardiomyopathy merit further investigation to determine whether DNA methylation changes play a causative role in the progression of heart failure.     

长春瑞滨联合卡铂化疗治疗晚期肺癌的临床观察

目的:探讨长春瑞滨联合卡铂化疗对晚期肺癌的治疗效果和安全性。方法:选取我院肿瘤科收治的晚期肺癌患者60例,根据不同治疗方案分为实验组与对照组,每组各30例患者。比较两组患者治疗前后的CEA、CA50、CYFRA21-1水平、白细胞计数及T淋巴细胞亚群CD4+、CD8+水平的变化。结果:治疗前,两组患者的CEA、CA50、CYFRA21-1水平、白细胞计数及T淋巴细胞亚群CD4+、CD8+水平比较均无统计学差异(P0.05)。治疗后,实验组的CEA、CA50、CYFRA21-1、CD8+水平均明显低于对照组,而白细胞计数及T淋巴细胞亚群CD4+、CD4+/CD8+水平均显著高于对照组,差异均具有统计学意义(P0.05)。结论:与放疗相比,长春瑞滨联合卡铂化疗能显著提高晚期肺癌的疗效,且不降低患者的细胞免疫。     

Modeling the Repertoire of True Tumor-Specific MHC I Epitopes in a Human Tumor

DNA replication has a finite measurable error rate, net of repair, in all cells. Clonal proliferation of cancer cells leads therefore to accumulation of random mutations. A proportion of these mutational events can create new immunogenic epitopes that, if processed and presented by an MHC allele, may be recognized by the adaptive immune system. Here, we use probability theory to analyze the mutational and epitope composition of a tumor mass in successive division cycles and create a double Pölya model for calculating the number of truly tumor-specific MHC I epitopes in a human tumor. We deduce that depending upon tumor size, the degree of genomic instability and the degree of death within a tumor, human tumors have several tens to low hundreds of new, truly tumor-specific epitopes. Parenthetically, cancer stem cells, due to the asymmetry in their proliferative properties, shall harbor significantly fewer mutations, and therefore significantly fewer immunogenic epitopes. As the overwhelming majority of the mutations in cancer cells are unrelated to malignancy, the mutation-generated epitopes shall be specific for each individual tumor, and constitute the antigenic fingerprint of each tumor. These calculations highlight the benefits for personalization of immunotherapy of human cancer, and in view of the substantial pre-existing antigenic repertoire of tumors, emphasize the enormous potential of therapies that modulate the anti-cancer immune response by liberating it from inhibitory influences.     

HPV感染致HeLa细胞肿瘤标志物水平的变化

人乳头瘤病毒(Human papillomavirus,HPV)是宫颈癌的重要病因学因素.肿瘤标志物能评估肿瘤的发生与发展,宫颈癌相关肿瘤标志物包括鳞状细胞癌(Squamous cell carcinoma,SCC)相关抗原、癌胚抗原(Carcino-embryonic atigen,CEA)、糖类抗原(Carbohydrate antigen,CA)125和 CA19-9.为探索不同类型 HPV 对宫颈癌细胞(HeLa细胞)肿瘤标志物的影响,为宫颈癌早期诊断及病情评估提供理论参考,本研究收集2019年1月至6月在西南医科大学附属医院妇科接受宫颈检查的受试者宫颈上皮细胞样本,提取不同种类HPV接种于HeLa细胞.根据所接种的HPV类型不同,将HeLa细胞分为高危型组、中危型组、低危型组以及阴性组.以Q-PCR及Western Blot方法检测SCC及CEA基因与蛋白的相对表达量,以ELISA法检测CA125及CA19-9表达水平.Q-PCR结果显示:与阴性组相比,高危组、中危组和低危组三组SCC与CEA基因相对表达量明显增高(P＜0.05);三组间两两比较,均有统计学差异(P＜0.05),其中高危组表达量＞中危组＞低危组;Western Blot结果可见:与阴性组相比,高危组、中危组和低危组三组SCC与CEA蛋白相对表达量明显增高(P＜0.05);三组间两两比较,均有统计学差异(P＜0.05),其中高危组表达量＞中危组＞低危组;ELISA检测结果可见:与阴性组相比,高危组、中危组和低危组三组CA125与CA19-9其表达量明显增高(P＜0.05);三组间两两比较,均有统计学差异(P＜0.05),其中高危组表达量＞中危组＞低危组.本研究提示,不同致病类型HPV与肿瘤标志物表达呈相关性,HPV危险程度越高,其肿瘤标志物表达量越大.     

SPARC Expression Correlates with Tumor Response to Albumin-Bound Paclitaxel in Head and Neck Cancer Patients

SPARC up-regulation is a poor prognostic factor in head and neck cancer. It was hypothesized that because of a SPARC-albumin interaction, tumoral SPARC facilitates the accumulation of albumin in the tumor and increases the effectiveness of albumin-bound paclitaxel (nab-paclitaxel). This hypothesis was tested by correlating the response to nab-paclitaxel and SPARC tumor expression in a retrospective analysis of a 60-patient clinical study of nab-paclitaxel as monotherapy against head and neck cancer. Sixteen tumor specimens were available for analysis. There were 11 responders (CR/PR) and 5 nonresponders (SD/PD) among the 16 nab-paclitaxel-treated patients (12/16 SPARC-positive, 75%). Response to nab-paclitaxel was higher for SPARC-positive patients (10/12, 83%) than SPARC-negative patients (1/4, 25%). The SPARC-negative patients exhibited significantly lower response than the overall response rate among all 60 patients (1/4, 25% vs 45/60, 75%). Although preliminary, data are supportive of the hypothesis that SPARC overexpression may correlate with response to nab-paclitaxel. If confirmed in larger studies, treatment with nab-paclitaxel may convert a poor prognosis SPARC-positive patient population into a group with better clinical outcomes.     

血管内皮标记物及血管内皮生长因子在胸水细胞中的表达和意义

探讨CD31、CD34、CD105及VEGF在胸水中的表达.应用免疫细胞化学染色,免疫荧光染色,Western-blot技术检测200例非小细胞肺癌患者胸水、30例增生胸水和20例炎性胸水中CD31、CD34、CD105及VEGF的表达.CD31、CD34、CD105及VEGF在非小细胞肺癌胸水中的表达量明显高于增生和炎性胸水表达(P＜0.05).非小细胞肺癌胸水患者CD31、CD34、CD105及VEGF高表达,并且在存在着肿瘤细胞血管样管型结构中表达量明显高于未发现肿瘤细胞血管样管型的胸腔积液.检测CD31、CD34、CD105及VEGF在胸腔积液中的表达情况可能对判断患者的预后有一定价值.     

Endothelial Heparan Sulfate 6-O-Sulfation Levels Regulate Angiogenic Responses of Endothelial Cells to Fibroblast Growth Factor 2 and Vascular Endothelial Growth Factor

Fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor 165 (VEGF₁₆₅) are potent pro-angiogenic growth factors that play a pivotal role in tumor angiogenesis. The activity of these growth factors is regulated by heparan sulfate (HS), which is essential for the formation of FGF2/FGF receptor (FGFR) and VEGF₁₆₅/VEGF receptor signaling complexes. However, the structural characteristics of HS that determine activation or inhibition of such complexes are only partially defined. Here we show that ovarian tumor endothelium displays high levels of HS sequences that harbor glucosamine 6-O-sulfates when compared with normal ovarian vasculature where these sequences are also detected in perivascular area. Reduced HS 6-O-sulfotransferase 1 (HS6ST-1) or 6-O-sulfotransferase 2 (HS6ST-2) expression in endothelial cells impacts upon the prevalence of HS 6-O-sulfate moieties in HS sequences, which consist of repeating short, highly sulfated S domains interspersed by transitional N-acetylated/N-sulfated domains. 1–40% reduction in 6-O-sulfates significantly compromises FGF2- and VEGF₁₆₅-induced endothelial cell sprouting and tube formation in vitro and FGF2-dependent angiogenesis in vivo. Moreover, HS on wild-type neighboring endothelial or smooth muscle cells fails to restore endothelial cell sprouting and tube formation. The affinity of FGF2 for HS with reduced 6-O-sulfation is preserved, although FGFR1 activation is inhibited correlating with reduced receptor internalization. These data show that 6-O-sulfate moieties in endothelial HS are of major importance in regulating FGF2- and VEGF₁₆₅-dependent endothelial cell functions in vitro and in vivo and highlight HS6ST-1 and HS6ST-2 as potential targets of novel antiangiogenic agents.     

Crotepoxide Chemosensitizes Tumor Cells through Inhibition of Expression of Proliferation,Invasion, and Angiogenic Proteins Linked to Proinflammatory Pathway

Crotepoxide (a substituted cyclohexane diepoxide), isolated from Kaempferia pulchra (peacock ginger), although linked to antitumor and anti-inflammatory activities, the mechanism by which it exhibits these activities, is not yet understood. Because nuclear factor κB (NF-κB) plays a critical role in these signaling pathways, we investigated the effects of crotepoxide on NF-κB-mediated cellular responses in human cancer cells. We found that crotepoxide potentiated tumor necrosis factor (TNF), and chemotherapeutic agents induced apoptosis and inhibited the expression of NF-κB-regulated gene products involved in anti-apoptosis (Bcl-2, Bcl-xL, IAP1,² MCl-1, survivin, and TRAF1), apoptosis (Bax, Bid), inflammation (COX-2), proliferation (cyclin D1 and c-myc), invasion (ICAM-1 and MMP-9), and angiogenesis (VEGF). We also found that crotepoxide inhibited both inducible and constitutive NF-κB activation. Crotepoxide inhibition of NF-κB was not inducer-specific; it inhibited NF-κB activation induced by TNF, phorbol 12-myristate 13-acetate, lipopolysaccharide, and cigarette smoke. Crotepoxide suppression of NF-κB was not cell type-specific because NF-κB activation was inhibited in myeloid, leukemia, and epithelial cells. Furthermore, we found that crotepoxide inhibited TAK1 activation, which led to suppression of IκBα kinase, abrogation of IκBα phosphorylation and degradation, nuclear translocation of p65, and suppression of NF-κB-dependent reporter gene expression. Overall, our results indicate that crotepoxide sensitizes tumor cells to cytokines and chemotherapeutic agents through inhibition of NF-κB and NF-κB-regulated gene products, and this may provide the molecular basis for crotepoxide ability to suppress inflammation and carcinogenesis.     

Permeability of the Ehrlich Ascites Tumor Cell to Water

The osmotic permeability coefficient for water has been measured for the Ehrlich mouse ascites tumor cell. Measurements were made of the rate of cell shrinkage in hyperosmotic solutions of NaCI, a functionally impermeable solute. During the first 9 months of weekly serial transplantation the mean was 6.4 µ³/µ³/atm. ± 0.8 (S.E.). By the end of the 2nd year the permeability coefficient was much lower and averaged 1.6 ± 0.09. There were no significant differences in the volume of the tumor cells which could explain the discrepancy on the basis of a change in the volume to surface area ratio. Studies of the effect of temperature were done and Eyring's theory of absolute reaction rates was applied to the data. The apparent energy of activation was 9.6 kcal./mol and ΔS‡ was 39.1 entropy units. The thermodynamic data are twice as high as data reported by Wang for self-diffusion and viscous properties of water. Two alternate explanations have been advanced based on the pore hypothesis of membrane permeability. One explains the thermodynamic data from a change in the A'/Δx available for water movement; the other assumes A'/Δx constant and bases the results on the interaction of water dipoles with each other and the membrane.     

Low Levels of the Reverse Transactivator Fail to Induce Target Transgene Expression in Vascular Smooth Muscle Cells

Hutchinson-Gilford progeria syndrome (HGPS) is a genetic disease with multiple features that are suggestive of premature aging. Most patients with HGPS carry a mutation on one of their copies of the LMNA gene. The LMNA gene encodes the lamin A and lamin C proteins, which are the major proteins of the nuclear lamina. The organs of the cardiovascular system are amongst those that are most severely affected in HGPS, undergoing a progressive depletion of vascular smooth muscle cells, and most children with HGPS die in their early teens from cardio-vascular disease and other complications from atherosclerosis. In this study, we developed a transgenic mouse model based on the tet-ON system to increase the understanding of the molecular mechanisms leading to the most lethal aspect of HGPS. To induce the expression of the most common HGPS mutation, LMNA c.1824C>T; p.G608G, in the vascular smooth muscle cells of the aortic arch and thoracic aorta, we used the previously described reverse tetracycline-controlled transactivator, sm22α-rtTA. However, the expression of the reverse sm22α-transactivator was barely detectable in the arteries, and this low level of expression was not sufficient to induce the expression of the target human lamin A minigene. The results from this study are important because they suggest caution during the use of previously functional transgenic animal models and emphasize the importance of assessing transgene expression over time.     

Identification of Tumor Endothelial Cells with High Aldehyde Dehydrogenase Activity and a Highly Angiogenic Phenotype

Tumor blood vessels play an important role in tumor progression and metastasis. It has been reported that tumor endothelial cells (TECs) exhibit highly angiogenic phenotypes compared with those of normal endothelial cells (NECs). TECs show higher proliferative and migratory abilities than those NECs, together with upregulation of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2). Furthermore, compared with NECs, stem cell markers such as Sca-1, CD90, and multidrug resistance 1 are upregulated in TECs, suggesting that stem-like cells exist in tumor blood vessels. In this study, to reveal the biological role of stem-like TECs, we analyzed expression of the stem cell marker aldehyde dehydrogenase (ALDH) in TECs and characterized ALDH^high TECs. TECs and NECs were isolated from melanoma-xenografted nude mice and normal dermis, respectively. ALDH mRNA expression and activity were higher in TECs than those in NECs. Next, ALDH^high/low TECs were isolated by fluorescence-activated cell sorting to compare their characteristics. Compared with ALDH^low TECs, ALDH^high TECs formed more tubes on Matrigel-coated plates and sustained the tubular networks longer. Furthermore, VEGFR2 expression was higher in ALDH^high TECs than that in ALDH^low TECs. In addition, ALDH was expressed in the tumor blood vessels of in vivo mouse models of melanoma and oral carcinoma, but not in normal blood vessels. These findings indicate that ALDH^high TECs exhibit an angiogenic phenotype. Stem-like TECs may have an essential role in tumor angiogenesis.     

Decreased Expression of PTPN12 Correlates with Tumor Recurrence and Poor Survival of Patients with Hepatocellular Carcinoma

Background

Protein tyrosine phosphatase non-receptor type 12 (PTPN12), has been identified as a potent tumor suppressor in human cancers and a critical regulator of cell adhesion and migration. However, the PTPN12 expression and its prognostic significance in HCC have not been well elucidated.

Methodology/Principal Findings

In this study, tissue microarray-based immunohistochemistry (IHC) was investigated in an HCC cohort with adjacent liver tissues as controls. The resulting data were analyzed using receiver operating characteristic curves, Spearman''s rank correlation, Kaplan-Meier plots and Cox proportional hazards regression modeling. Our results showed that decreased expression of PTPN12 was more frequently observed in HCC tissues compared to the adjacent non-tumorous liver tissues. Further correlation analyses indicated that the decreased PTPN12 expression was closely correlated with tumor recurrence (P = 0.015). Univariate analysis showed a significant association between decreased expression of PTPN12 and adverse cancer-specific survival and recurrence-free survival (P<0.001). In different subsets of overall patients, PTPN12 expression was also a prognostic indicator in patients with stage I/II or stage III/IV (P<0.05). Importantly, multivariate analysis (P<0.05) identified PTPN12 expression in HCC as an independent prognostic factor.

Conclusions/Significance

Our findings provide a basis for the concept that PTPN12 protein expression is frequently decreased or lost in human HCC tissues and that decreased PTPN12 expression may represent an acquired recurrence phenotype of HCC and that PTPN12 expression may act as a biomarker of prognosis for patients with HCC.     

Influence of Human Seminal Proteases on Vascular Permeability

HUMAN seminal fluid contains many enzymes among which are hydrolases capable of splitting proteins and arginine esters¹. The seminal proteases and esterases can be separated into several fractions by DEAE-cellulose chromatography and ‘Sephadex G’ gel filtration. Two of the fractions have enzymatic characteristics similar to those of chymotrypsin and four different fractions hydrolyse arginine esters². The hydrolysis of arginine esters has been found to occur in the male accessory sex glands of some laboratory rodents³. From the coagulating gland of the guinea-pig an arginine esterase was separated by electrophoresis, which was able to increase vascular permeability⁴. Some of the characteristics of the human seminal arginine esterases resemble those of kallikreins⁵ in that the latter can, for example, increase vascular permeability⁶. We have therefore investigated the effect of several protease fractions of the human seminal fluid on the vascular permeability.     

High Bone Sialoprotein (BSP) Expression Correlates with Increased Tumor Grade and Predicts a Poorer Prognosis of High-Grade Glioma Patients

Objectives

To investigate the expression and prognostic value of bone sialoprotein (BSP) in glioma patients.

Methods

We determined the expression of BSP using real-time RT-PCR and immunohistochemistry in tissue microarrays containing 15 normal brain and 270 glioma samples. Cumulative survival was calculated by the Kaplan-Meier method and analyzed by the log-rank test. Univariate and multivariate analyses were performed by the stepwise forward Cox regression model.

Results

Both BSP mRNA and protein levels were significantly elevated in high-grade glioma tissues compared with those of normal brain and low-grade glioma tissues, and BSP expression positively correlated with tumor grade (P<0.001). Univariate and multivariate analysis showed high BSP expression was an independent prognostic factor for a shorter progression-free survival (PFS) and overall survival (OS) in both grade III and grade IV glioma patients [hazard ratio (HR) = 2.549 and 3.154 for grade III glioma, and HR = 1.637 and 1.574 for grade IV glioma, respectively]. Patients with low BSP expression had a significantly longer median OS and PFS than those with high BSP expression. Small extent of resection and lineage of astrocyte served as independent risk factors of both shorter PFS and OS in grade III glioma patients; GBM patients without O⁶-methylguanine (O⁶-meG) DNA methyltransferase (MGMT) methylation and Karnofsky performance score (KPS) less than 70 points were related to poor prognosis. Lack of radiotherapy related to shorter OS but not affect PFS in both grade III and grade IV glioma patients.

Conclusion

High BSP expression occurs in a significant subset of high-grade glioma patients and predicts a poorer outcome. The study identifies a potentially useful molecular marker for the categorization and targeted therapy of gliomas.