Microvessel Density Values Research Articles

Prognostic Value of Microvessel Density and p53 Expression on the Locoregional Metastasis and Survival of the Patients With Head and Neck Squamous Cell Carcinoma

Cancer cells need to develop microvessels in order to grow and to establish metastatic foci. A role for the p53 protein in the regulation of the angiogenic process is suggested. This study aimed to investigate the relationship between immunohistochemical expression of microvessel density (MVD), measured by CD31 staining, and p53 protein with clinicopathologic factors, and survival in head and neck squamous cell carcinoma (n=70). Tumor angiogenesis was estimated by determining MVD in areas with the highest number of stained microvessels (hot spots). Clinicopathologic factors and immunohistochemical data were evaluated by χ statistical test and were submitted to binary logistic regression to analyze the risk of presence of lymph node metastasis. Factors that might predict survival were investigated using Cox proportional hazards tests. Differences were considered statistically significant when P<0.05. The percentage of p53-positive cells showed no association with clinicopathologic parameters and MVD. Patients with locoregional metastasis presented statistically significant higher MVD (P=0.043). Individuals presenting head and neck squamous cell carcinoma in posterior sites (P=0.022; OR=3.644) and higher MVD (P=0.039; OR=3.247) had a significant increase in risk of metastasis occurrence. Multivariate analysis showed that presence of lymph node metastasis was statistically significant for overall survival of head and neck carcinoma patients (P=0.006; OR =2.917). The present data suggest that MVD represents a promising diagnostic tool to identify individuals with increased risk for the development of metastatic disease, which is very indicative of poor prognosis.

Antitumor effects of 32P-chromic-poly (L-lactide) brachytherapy in nude mice with human prostate cancer

The aim of the present study was to investigate the antitumor effects and tissue distribution of 32P-chromic-poly (L-lactide) (32P-CP-PLLA) in nude mice with human prostate cancer. Tumor models were obtained by transplantation of PC-3M tumor cells into male BALB/c nude mice. Animals were randomly divided into control, 32P-chromic phosphate (32P-CP) colloid and 32P-CP-PLLA groups (all n=20). A series of indices were investigated, including apoptosis of tumor cells, rate of apoptosis, expression of caspase 3 and 8, biodistribution and intratumoral concentration of 32P-CP-PLLA, intensity of radioactivity, tumor volume and microvessel density (MVD). Highly concentrated radioactivity of 32P-CP-PLLA in the tumor mass was detected by single photon emission computed tomography (SPECT) scanning. The residual activities of the 32P-CP-PLLA and 32P-CP colloid groups were 3.02±0.32 and 1.76±0.31 MBq, respectively, on day 14 following treatment. The tumor inhibition rates were 67.24±3.55 and 55.92±7.65%, respectively (P<0.01). Necrotic changes, in conjunction with apoptosis, were observed in the treatment group. MVD values for the 32P-CP-PLLA and 32P-CP colloid groups were 28.24±10.07 and 36.15±11.06, respectively. 32P-CP-PLLA showed an excellent capacity for killing tumor cells, inducing apoptosis and inhibiting angiogenesis.

Intratumoral vessel density as prognostic factors in head and neck squamous cell carcinoma: A meta‐analysis of literature

The purpose of this study was to comprehensively and quantitatively summarize the prognostic value of microvessel density (MVD) and lymphatic vessel density (LVD) for the survival of patients with head and neck squamous cell carcinoma (HNSCC). Relevant literatures were identified using Medline and EMBASE. A meta-analysis was performed to clarify the prognostic role of MVD and LVD. A total of 18 articles (13 for MVD and 5 for LVD; n = 1279 and n = 408) were pooled for the global meta-analysis. The mortality was 1.23-fold higher for patients whose MVD counts were above the cutoff (risk ratio [RR], 1.23; 95% confidence interval [CI], 0.99-1.52), compared with those below it, and this result was on a borderline (p = .06). The mortality of patients with high LVD counts was 2.07-fold higher (RR, 2.07; 95% CI, 1.16-3.71; p = .01). High counts of LVD, not MVD, are associated with worse 5-year overall survival (OS) in patients with HNSCC.

Tumor angiogenesis, macrophages and mast cell microdensities in endometrioid endometrial carcinoma

The present study aimed to observe and compare the values of microvessel density (MVD), mast cell microdensity (McMD) and macrophage microdensity (MphMD) in intratumoral areas compared with the advancing edges, and to assess any correlations between these values and the degree and stage of the neoplasia. The cases of 52 patients who were diagnosed with endometrial carcinoma between 2003 and 2011 were analyzed, the majority of which were in the first stage of the disease (44 cases). Double sequential immunohistochemistry and the hot-spot counting method were used to assess the MVD (CD105+ MVD), McMD [tryptase+ (Try+) McMD] and MphMD (CD68+ MphMD) densities. The χ2 test, paired Student’s t-test and the Pearson correlation index were used to assess the significance of the results. A weak correlation was observed at the advancing edge only, between CD105+ MVD and Try+ McMD (P=0.039). No significant differences were identified in the analysis of CD105+ MVD, Try+ McMD and CD68+ MphMD, but wide variations in their distribution were observed. Depending on the tumor stage, CD105+ MVD exhibited an intratumoral, indirect correlation with Try+ McMD for stage IA (P=0.026) and II (P=0.013) tumors. CD105+ MVD presented an indirect correlation with CD68+ MphMD in stage IB tumors (P=0.016) and at the advancing edge for well-differentiated tumors (P=0.027). An analysis of the correlation between CD68+ MphMD and Try+ McMD indicated that the intratumoral levels of CD68+ MphMD were directly proportional with the Try+ McMD values in well-differentiated (P=0.005) and stage II (P=0.012) tumors, while at the front of the invasion, this correlation was indirect (P=0.010) in stage II tumors. In endometrioid endometrial carcinoma (EEC), angiogenesis is at its most active at the advancing edge of the tumor, where mast cells play a pro-angiogenic role.

WE-G-103-01: Dual-Energy Temporal Study of Contrast-Enhanced Digital Mammography in 20 Patients: The Use of a Pixel-By-Pixel Weighting Formalism for the Subtraction

Purpose: To study the possible correlation between iodine uptake in contrast-enhanced digital mammography images and microvessel density (MVD) in breast lesions. Methods: 20 BIRADS 4–5 patients were included. Mask images were acquired with low-and high-energy spectra from a SenographeDS. Iodine-based contrast medium (CM) was mechanically injected and high-energy CM images acquired between 1 and 5 minutes after injection. A biopsy was obtained after the images and specific biomarkers for newly-formed blood and lymphatic microvessels were applied; MVD was later evaluated in microscope fields. The subtraction combined dual-energy and temporal modalities. LE masks were subtracted from weighted CM images, and weight factor was a matrix obtained from the masks, containing pixel-by-pixel anatomical and radiological information. Iodine uptake in subtracted temporal series was quantified by contrast between lesion and normal glandular tissue. Alternative metrics for contrast quantification were evaluated. Contrast was transformed into iodine mass-thickness using calibrated samples. Results: 12 cases were malignant and 8, benign. The matrix-based subtraction formalism severely reduced anatomic noise in resulting images, compared with alternative techniques based on mean pixel values within regions-of-interest. Five types of time-intensity curves were identified, qualitatively similar to MRI. If iodine uptake in the lesion was evaluated, most frequent curve was plateau type, for cancer and malignant cases. The normalized Weber contrast indicated a frequency distribution with zero-contrast cases for benign lesions and most-frequent curve type washout for cancer. No correlation was found between contrast indicators and MVD. Blood and lymphatic MVD were correlated (r=0.94, p<0.05) and mean blood MVD values in cancer were about twice those in benign cases. Conclusion: This subtraction formalism, previously validated in non-homogeneous phantom images, adds quantitative features to the resulting clinical images. The statistical distribution of contrast curves for cancer and benign cases seems to be the most relevant parameter with a possible diagnostic value. Funding: DGAPA-UNAM Grants 102610 and 105813; Conacyt Grant Salud 2009-01-112374. No conflict of interest.

Knockdown of NF-E2-related factor 2 inhibits the proliferation and growth of U251MG human glioma cells in a mouse xenograft model

NF-E2-related factor 2 (Nrf2) is a pivotal transcription factor of cellular responses to oxidative stress and recent evidence suggests that Nrf2 plays an important role in cancer pathobiology. However, the underlying mechanism has yet to be elucidated, particularly in glioma. In the present study, we investigated the role of Nrf2 in the clinical prognosis, cell proliferation and tumor growth of human glioblastoma multiforme (GBM). We detected overexpression of Nrf2 protein levels in GBM compared to normal brain tissues. Notably, higher protein levels of Nrf2 were significantly associated with poorer overall survival and 1-year survival for GBM patients. Furthermore, we constructed the plasmid Si-Nrf2 and transduced it into U251MG cells to downregulate the expression of Nrf2 and established stable Nrf2 knockdown cells. The downregulation of Nrf2 suppressed cell proliferation in vitro and tumor growth in mouse xenograft models. We performed immunohistochemistry staining to detect the protein levels of Nrf2, Ki-67, caspase-3 and CD31 in the xenograft tumors and found that the expression levels of Nrf2 and Ki-67 were much lower in the Si-Nrf2 group compared to the Si-control group. In addition, the number of caspase-3-positive cells was significantly increased in the Si-Nrf2 group. By analysis of microvessel density (MVD) assessed by CD31, the MVD value in the Si-Nrf2 group decreased significantly compared to the Si-control group. These findings indicate that the knockdown of Nrf2 may suppress tumor growth by inhibiting cell proliferation, increasing cell apoptosis and inhibiting angiogenesis. These results highlight the potential of Nrf2 as a candidate molecular target to control GBM cell proliferation and tumor growth.

Prognostic value of MVD, LVD and vascular invasion in lymph node-negative colon cancer.

To clarify the influence of microvessel density (MVD), lymphangiogenesis (LVD), and vascular invasion on prognosis in lymph node-negative colon cancer. We performed immunohistochemical analysis from 152 Duke's B colon carcinomas, CD34 and LYVE-1 antibodies. Carcinomas were graded as low or high grade. χ2 test was used to examine their relationships and correlations with clinicopathological parameters. Survival time was analyzed and the differences between groups were assessed. A statistically significantly correlation was found between increasing MVD with age >60 years, tumor size >4cm, and poor tumor differentiation (χ2=40.018, p<0.001). The increase in MVD was associated with shorter DFS (p<0.001) and shorter OS in patients with colon cancer (p<0.001). LVD was statistically significantly associated with increasing the number of newly created blood vessels (χ2=96.6, p<0.001), low degree of tumor differentiation (χ2=96.6, p<0.001), and vascular invasion (χ2=51.8, p<0.001) in colon cancer. Log rank analysis showed that positive staining for MVD and LVD, high histological grade, vascular invasion, male gender, and age >60 years were connected with shorter survival of patients with Dukes B colon cancer 45 vs. 100 months (p=0.016 to <0.001). Positive expression MVD and LVD was significantly correlation with survival time and with high tumor grade and vascular invasion in patients with Dukes B colon cancer. The correlation of MVD and LVD with vascular invasion in Dukes B colon cancer indicates the need for further confirmation as a possible predictive marker.

Abstract 369: Role of bone marrow angiogenesis in myeloma and its precursor disease: a prospective clinical trial.

Abstract Background: Based on bone marrow (BM) biopsies, increased angiogenesis has been reported in multiple myeloma (MM) and smoldering multiple myeloma (SMM) patients compared with individuals with monoclonal gammopathy of undetermined significance (MGUS). In this prospective clinical trial open for MGUS, SMM and MM patients, we conducted BM biopsies and used immunohistochemistry to define microvessel density (MVD) in every patient; results were compared to results obtained from dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and multiplex ELISA-based serum angiogenic cytokine assays. Patients and Methods: We included 10 MGUS, 10 SMM and 10 MM patients. We performed DCE-MRI of entire lumbosacral spine to compare exchange rate constants (contrast agent transit from the extravascular compartment to the intravascular compartment, Kep; movement of contrast agent from plasma to extravascular extracellular space, Ktrans) with bone marrow biopsies immunostained with CD34 as measures of MVD, and serum angiogenic markers By ELISA to include EGF, Ang2, GCSF, BMP9, endoglin, Leptin, Follistatin, IL8, HGF, HBEGF, PLGF, VEGFC, VEGFD, FGF2, VEGFA. The association among continous parameters was determined by Spearman rank correlation. Trends in continuous parameters according to ordered categorical parameter were determined by Jonckheere-Terpstra test for trend. The parameters between two groups were compared using an exact Wilcoxon rank sum test. Results: Both MVD and Kep increased along the myeloma spectrum (MGUS &amp;lt; SMM &amp;lt; MM). MVD and Kep were strongly correlated (r=0.93 and p=0.002). Ktrans was weakly to moderately well correlated with MVD (r=0.43 p=0.03). Higher Kep values were seen in MM/SMM vs. MGUS patients (median 7.1 vs. 3.9; p=0.08). Similarly, higher MVD values were seen in MM/SMM patients versus MGUS (median 20 vs. 15; p=0.01). As regards serum angiogenic markers, levels of HGF (r=0.45; p=0.02), Ang2(r=0.37 and p=0.06), and VEGFD (r=-0.35 and p=0.07), correlated with Kep. The levels of Ang2 (p=0.02), GCSF (p=0.06), follistatin (p=0.06), HGF (p=0.01), and VEGFA (p=0.02), were elevated in MM/SMM patients in comparison to MGUS. Other angiogenic cytokines did not correlate with MVD or Kep. Conclusions: MVD and Kep (measured by DCE-MRI) were highly statistically correlated. MVD increased along the disease spectrum from MGUS to SMM to MM (p=0.008). Some, but not all, angiogenic biomarkers detected in peripheral blood were associated with increased vascularity in the bone marrow. Citation Format: Manisha Bhutani, Esther Mena, Irina Maric, Esther Tan, Neha Korde, Alexandra R. Berg, Alex R. Minter, Brendan M. Weiss, Liza Lindenberg, Baris Turkbey, Omer Aras, Seth Steinberg, Troy Kemp, Katherine R. Calvo, Peter L. Choyke, Karen Kurdziel, Ola Landgren. Role of bone marrow angiogenesis in myeloma and its precursor disease: a prospective clinical trial. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 369. doi:10.1158/1538-7445.AM2013-369

Vascular proliferation is associated with survival in pancreatic ductal adenocarcinoma

In pancreatic ductal adenocarcinoma (PDAC), the benefit of current chemotherapy and radiation therapy is very limited, even in radically resected patients. New treatment strategies, for example based on the inhibition of the tumour's blood supply, need to be explored. We have investigated angiogenesis markers and their associations with relapse and survival in 52 histologically confirmed cases of PDAC. Angiogenesis in the primary tumour was evaluated by microvessel density (MVD), vascular proliferation index (VPI) and the presence of glomeruloid microvascular proliferations (GMP). These features were analysed in the context of clinicopathological variables, KRAS mutation status, relapse location and survival. MVD (median 134 microvessels/mm(2) , range 88-177) and VPI (median 3.2%, range 1.6-4.9) were associated with larger tumour size and lymph node metastasis. MVD was also related to the occurrence of liver metastases. Both variables were associated with survival in univariate and multivariate analyses. GMPs were present in 32 (62%) of the cases. Patients who exhibited MVD and VPI values above median, and GMP positivity, had a median survival of only 4.2 months after surgery. In conclusion, the angiogenesis markers MVD and VPI have a significant impact on survival. By also including GMP, a subgroup of PDAC patients with particularly short survival could be identified.

Correlation of Microvessel Density with Nuclear Pleomorphism, Mitotic Count and Vascular Invasion in Breast and Prostate Cancers at Preclinical and Clinical Levels

Tumor angiogenesis correlates with recurrence and appears to be a prognostic factor for both breast and prostate cancers. In the present study, we aimed to investigate the correlation of microvessel density (MVD), a measure of angiogenesis, with nuclear pleomorphism, mitotic count, and vascular invasion in breast and prostate cancers at preclinical and clinical levels. Samples from xenograft tumors of luminal B breast cancer and prostate adenocarcinoma, established by BT-474 and PC-3 cell lines, respectively, and commensurate human paraffin-embedded blocks were obtained. To determine MVD, specimens were immunostained for CD-34. Nuclear pleomorphism, mitotic count, and vascular invasion were determined using hematoxylin and eosin (HandE)-stained slides. MVD showed significant correlations with nuclear pleomorphism (r=0.68, P=0.03) and vascular invasion (r=0.77, P=0.009) in breast cancer. In prostate cancer, MVD was significantly correlated with nuclear pleomorphism (r=0.75, P=0.013) and mitotic count (r=0.75, P=0.012). In the breast cancer xenograft model, a significant correlation was observed between MVD and vascular invasion (r=0.87, P=0.011). In the prostate cancer xenograft model, MVD was significantly correlated with all three parameters (nuclear pleomorphism, r=0.95, P=0.001; mitotic count, r=0.91, P=0.001; and vascular invasion, r=0.79, P=0.017; respectively). Our results demonstrate that MVD is correlated with nuclear pleomorphism, mitotic count, and vascular invasion at both preclinical and clinical levels. This study therefore supports the predictive value of MVD in breast and prostate cancers.

Necrosis Degree Displayed in Computed Tomography Images Correlated With Hypoxia and Angiogenesis in Breast Cancer

The objective of this study was to investigate the correlation between the degree of necrosis displayed in computed tomography (CT) image and the expression of hypoxic and angiogenesis biomarkers of breast cancer. Forty-four breast cancer cases were examined with CT before surgery. Tumor specimen expressions of glucose transporter 1 (Glut1), hypoxia-inducible factor 1α (HIF-1α), carbonic anhydrase IX (CA IX), vascular endothelial growth factor (VEGF), and CD34 (as a marker of vascular endothelial cells) were detected by immunohistochemistry. The expressions of Glut1 and CA IX were localized primarily to the edges of necrotic areas or intraduct surface; there was a strong correlation between HIF-1α-positive expression and CA IX-positive expression (P < 0.001), and higher Glut1 or CA IX expression grade was associated with lower microvessel density (MVD) value. In CT enhanced images, lower relative CT (rCT) values were associated with more significant necrosis in the tumor; rCT value correlated positively with MVD significantly (r = 0.319, P = 0.035), and higher Glut1 or CA IX expression grade correlated with lower rCT values (P = 0.001 and 0.003, respectively). Although high VEGF expression was significantly correlated with high HIF-1α expression (P < 0.001), there were no correlations between VEGF and MVD, and HIF-1α and MVD (P = 0.559 and 0.710, respectively), and the difference of the mean rCT value between high VEGF or HIF-1α expression group and low VEGF or HIF-1α expression group was not significant. In breast cancer tissues, Glut1 and CA IX are key hypoxia biomarkers. Importantly, detection of necrosis in breast cancer tissue via CT enhanced imaging may prognosticate hypoxia and angiogenesis status and help to determine treatment plan of advanced breast cancer.

Correlation between Microvessel Density and CT Value of the Keratocystic Odontogenic Tumor in the Mandible

Correlation between Microvessel Density and CT Value of the Keratocystic Odontogenic Tumor in the Mandible

Dynamic Contrast-Enhanced MR Imaging in a Phase Ⅱ Study on Neoadjuvant Chemotherapy Combining Rh-Endostatin with Docetaxel and Epirubicin for Locally Advanced Breast Cancer

Background: Anti-angiogenesis is a promising therapeutic strategy for locally advanced breast cancer. We performed this phase II trial to evaluate the anti-angiogenesis and anti-tumor effect of rh-endostatin combined with docetaxel and epirubicin in patients with locally advanced breast cancer by dynamic contrast-enhanced magnetic resonance imaging in 70 previously untreated locally advanced breast cancer patients.Methods: The study population was randomly assigned to neoadjuvant chemotherapy with docetaxel and epirubicin (neoadjuvant chemotherapy group) or neoadjuvant chemotherapy combining rh-endostatin with docetaxel and epirubicin (neoadjuvant chemotherapy+rh-endostatin group). The anti-angiogenic and anti-tumor effects of both regimens were evaluated by serial dynamic contrast-enhanced magnetic resonance imaging and microvessel density measurements after final surgery.Results: The results suggested a higher clinical objective response (90.9% vs. 67.7%, P = 0.021) and greater reductions in tumor size (67.2% vs. 55.9%, P = 0.000), Ki-67 proliferation index (32.79% vs. 12.47%, P = 0.000), tumor signal enhanced ratio (64% vs. 48%, P = 0.018), and Ktrans (67% vs. 39%, P = 0.026) in neoadjuvant chemotherapy+rh-endostatin group than those in neoadjuvant chemotherapy group. In addition, the microvessel density value in the neoadjuvant chemotherapy+rh-endostatin group was significantly lower than in the neoadjuvant chemotherapy group (18.67 ± 6.53 vs. 36.05 ± 9.64, P = 0.000). Moreover, the microvessel density value was significantly correlated with Ktrans after neoadjuvant chemotherapy+rh-endostatin treatment (r=0.88, P = 0.00).Conclusions: The neoadjuvant chemotherapy+rh-endostatin treatment significantly repressed angiogenesis in locally advanced breast cancer and synergistically enhanced the anti-tumor effect of neoadjuvant chemotherapy. Serial dynamic contrast-enhanced magnetic resonance imaging data including reductions in tumor size and Ktrans, could provide non-invasive evaluation for chemotherapeutic efficacy and, consequently, optimization of individual chemotherapy for locally advanced breast cancer patients.

Microvessel density and p53 mutations in advanced-stage epithelial ovarian cancer

We planned to determine the relationship between angiogenesis and p53 mutational status in advanced-stage epithelial ovarian cancer. Using 190 tumor samples from patients with stage III and IV ovarian cancer we performed p53 sequencing, immunohistochemistry, and CD31 microvessel density (MVD) determination. MVD was elevated in tumors with p53 null mutations compared to p53 missense mutation or no mutation. Disease recurrence was increased with higher MVD in both unadjusted and adjusted analyses. In adjusted analysis, p53 null mutation was associated with increased recurrence and worse overall survival. Worse overall survival and increased recurrence risk were also associated with the combination of CD31 MVD values >25 vessels/HPF and any p53 mutation. P53 mutation status and MVD may have prognostic significance in patients with advanced-stage ovarian cancer. Tumors with p53 null mutations are likely to be more vascular, contributing to decreased survival and increased recurrence probability.

Effects of combining Taxol and cyclooxygenase inhibitors on the angiogenesis and apoptosis in human ovarian cancer xenografts

The present study aimed to investigate the combined effects of Taxol and cyclooxygenase (COX) inhibitors on angiogenesis and cell apoptosis of SKOV-3 human ovarian carcinoma cell xenograft-bearing mice. The experiments were continued for 28 days. Animals were treated with 3 mg/kg SC-560 (a COX-1-selective inhibitor) alone, 100 mg/kg celecoxib (a COX-2-selective inhibitor) alone or SC-560/celecoxib by gavage twice a day, 20 mg/kg Taxol alone intraperitoneally once a week or in combination with SC-560 or celecoxib or SC-560/celecoxib/Taxol for three weeks. The mRNA levels of vascular endothelial growth factor (VEGF) was determined by reverse transcription-polymerase chain reaction (RT-PCR). The microvessel density (MVD) of ovarian carcinoma was determined by immunohistochemistry with anti-CD34 as the label. The apoptotic index was detected by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) method. The MVD value and apoptotic index in the SC-560/Taxol group were notably inhibited compared with the Taxol group (P<0.001). Moreover, the VEGF mRNA levels, MVD value and apoptotic index in the SC-560/Taxol group were significantly different from the celecoxib/Taxol group (P<0.05, P<0.05 and P<0.001, respectively). The present study demonstrated that SC-560 enhances the anti-angiogenic and pro-apoptotic effects of Taxol and these effects are better than with celecoxib.

Longitudinalin vivomonitoring of rodent glioma models through thinned skull using laser speckle contrast imaging

Laser speckle contrast imaging (LSCI) is a contrast agent free imaging technique suited for longitudinal assessment of vascular remodeling that accompanies brain tumor growth. We report the use of LSCI to monitor vascular changes in a rodent glioma model. Ten rats are inoculated with 9L gliosarcoma cells, and the angiogenic response is monitored five times over two weeks through a thinned skull imaging window. We are able to visualize neovascularization and measure the number of vessels per unit area to assess quantitatively the microvessel density (MVD). Spatial spread of MVD reveals regions of high MVD that may correspond to tumor location. Whole-field average MVD values increase with time in the tumor group but are fairly stable in the control groups. Statistical analysis shows significant differences in MVD values between the tumor group and both saline-receiving and unperturbed control groups over the two-week period (p<0.05). In conclusion, LSCI is suitable for investigation of tumor angiogenesis in rodent models. In addition, the statistical difference (p<0.02) between MVD values of the tumor (24.40 ± 1.41) and control groups (15.40 ± 1.60) on the 14th day after inoculation suggests a potential use of LSCI in the clinic in distinguishing tumor environments from normal vasculature.

Epigenetic silencing of Id4 identifies a glioblastoma subgroup with a better prognosis as a consequence of an inhibition of angiogenesis

Inhibitors of DNA binding/differentiation (Id1 to Id4) are a family of helix-loop-helix transcription factors, which are highly expressed during embryogenesis and at lower levels in mature tissues. Id4 plays an important role in neuronal stem cell differentiation, and its deregulation has been implicated in glial neoplasia. The methylation status of Id4 was analyzed by methylation-specific polymerase chain reaction (PCR) in 62 glioblastoma (GBM) cases and in 20 normal brain tissues. Methylation status of Id4 was confirmed by sequencing after subcloning and messenger RNA (mRNA) and protein expression. We also evaluated the mRNA expression of MGP (matrix GLA protein), TGF-β1 (transforming growth factor beta 1), and VEGF (vascular endothelial growth factor) by real-time PCR analysis. Clinical and histological assessment of tumor angiogenesis was performed by evaluating the relative enhancing tumor ratio on magnetic resonance imaging and microvessel density on von Willebrand factor-stained sections, respectively. The promoter of Id4 was methylated in 23 of 62 (37%) GBMs. In methylated GBMs, Id4 mRNA was significantly reduced, compared with unmethylated GBMs (P = .0002). A significant reduction of protein expression was detected in all hypermethylated cases. GBMs with methylated Id4 showed a significant reduction of MGP, TGF-β1, and VEGF mRNA expression and had significantly lower relative enhancing tumor ratio (P = .0108) and microvessel density (P = .0241) values with respect to unmethylated GBMs. Finally, Id4 methylation was significantly associated with a favorable clinical outcome (P = .0006). These data suggest that methylation of Id4 may be involved in the pathogenesis of GBM and in the resistance of this neoplasm to conventional treatment throughout MGP-mediated neoangiogenesis.

Inhibitory effect of compound cantharides capsule on the proliferation of xenografts of human hepatocellular carcinoma HepG(2)215 in mice

To investigate the inhibitory effect of compound cantharides capsules on the proliferation of xenografts of human hepatocellular carcinoma HepG(2215) in mice and their mechanism of action. One hundred healthy Balb/c mice (5-week old, male:female 1:1) were used in this study. Mouse models of human HepG(2215) hepatocarcinoma were established. The tumor-bearing mice were divided into five groups randomly. The control group A received daily intragastric administration of physiologic saline. The intervention groups B1, B2 and B3 were treated with compound cantharides capsule in a dose of 12.5 mg×kg(-1)×d(-1), 25 mg×kg(-1)×d(-1) and 37.5 mg×kg(-1)×d(-1), respectively, for 10 consecutive days. The group C had intraperitoneal injection of cyclophosphamide (25 mg×kg(-1)×d(-1)) for 10 consecutive days. The mice were sacrificed after the completion of administration. The tumors were taken out, the tumor volume was measured, the inhibitory rate of body weight was calculated, and the serum AFP concentration and the level of HBV DNA were determined. The survival of each group mice was analyzed. The levels of mRNA expression of apoptosis-related genes were assayed by quantitative RT-PCR. Apoptosis in the tumor cells was assayed with TUNEL staining. Flow cytometry was used to detect the levels of CD3(+), CD19(+), CD4(+) and CD8(+), and microvessel density (MVD) of the tumors was assessed by immunohistochemistry. After completion of the treatment, the inhibition rate of tumor growth of the groups B1, B2 and B3 was 29.8%, 38.7% and 48.1%, respectively, and that of the group C was 52.4%, with a significant difference among the groups (P < 0.05). The median survival time of the groups A, B1, B2, B3 and C was (30.0 ± 3.2) days, (49.0 ± 5.1) days, (50.0 ± 5.2) days, (57.5 ± 6.5) days and (49.0 ± 4.7) days, respectively. The median survival time of the group B3 was significantly longer than that of other groups (P < 0.05). The serum AFP level in the groups A, B1, B2, B3 and C was (492.7 ± 48.5) ng/ml, (281.2 ± 25.6) ng/ml, (194.3 ± 18.7) ng/ml, (170.1 ± 15.8) ng/ml and (138.7 ± 12.5) ng/ml, respectively, indicating that it was significantly inhibited in the group C. The inhibition rate of HBV DNA replication of the groups B1, B2, B3 and C was (46.0 ± 5.1)%, (65.5 ± 6.9)%, (81.3 ± 7.8)% and (19.5 ± 2.1)%, respectively, showing that compound cantharides capsules inhibited HBV DNA replication in a dose-dependent manner. The apoptosis rate of the groups A, B1, B2, B3 and C was (0.27 ± 0.03)%, (7.18 ± 2.12)%, (9.17 ± 2.42)%, (11.27 ± 3.03)% and (5.44 ± 2.45)%, respectively, and that of the group B3 was significantly higher than that of the groups A, B1, B2 and C (P < 0.05). The expression level of bax mRNA was significantly higher than that of the group C (P < 0.05). The drug could significantly decrease the bcl-2 mRNA expression level, more remarkably along with the increasing dose of cantharides, and it was significantly lower than that in the group C (P < 0.05). The levels of CD4(+), CD8(+), CD3(+) and CD19(+) were significantly higher than that in the groups A and C (P < 0.05). The value of MVD of the group B3 was significantly lower that that of groups A and C (P < 0.05). Compound cantharides capsules may inhibit the replication of HBV DNA in HepG(2215) cells, inducing apoptosis in the tumor cells, enhancing the immune function to inhibit the growth of liver cancer cells in mice, and significantly prolong the median survival time of tumor-bearing mice.

Increased Angiogenesis-associated Poor Outcome in Acute Lymphoblastic Leukemia

Angiogenesis in solid tumors is important for tumor growth, invasion, and metastasis. However, angiogenesis plays also an important role in hematological malignancies. We have analyzed the expression of vascular endothelial growth factor (VEGF) in the leukemic blast cells and microvessel density (MVD) in the bone marrow biopsy samples of the patients with acute lymphoblastic leukemia (ALL). Bone marrow MVD of the patients with ALL was significantly higher compared with normal controls and complete remission (P<0.001), but slightly lower than in patients with relapsed ALL (P>0.05). The bone marrow blast VEGF expression was significantly higher in newly diagnosed ALL patients, with predominant strong VEGF expression as compared with complete remission patients (who had negative or weak VEGF expression) (P<0.05), whereas initial values were slightly lower than in relapsed patients. There was a strong positive correlation between VEGF expression and MVD at presentation of ALL. Stronger expression of VEGF on blast cells indicates shorter overall survival in ALL. Furthermore, initial values of MVD had positive correlation with overall survival and leukemia-free survival (P=0.024 and P=0.017, respectively). Our data suggest that increased angiogenesis (confirmed by immunohistochemical expression of VEGF in leukemic blasts), and MVD may play an important role in the pathophysiology of ALL with prognostic implications. Thus, targeting VEGF pathway may bring the new approach for ALL treatment-using antiangiogenic drugs and tyrosine kinase inhibitors in combination with standard chemotherapy regimens.

Microvascularization and expression of VEGF and its receptors in recurring meningiomas: pathobiological data in favor of anti-angiogenic therapy approaches

We studied expression of molecules of the vascular endothelial growth factor (VEGF) pathway and its relation to vascularization, cell proliferation and patient outcome in recurring non-anaplastic meningioma. We studied 29 tumor specimens of 8 patients with recurring meningiomas and of 8 age- and gender-matched control patients with non-recurring meningiomas (including meningothelial, transitional, fibroblastic and atypical subtypes) using immunohistochemistry and in-situ hybridization. VEGF protein, VEGF-mRNA, VEGF receptor (VEGFR)-1 mRNA, VEGFR-2 mRNA and hypoxia-inducible factor (HIF)-1-α protein were expressed in 27/29 (93%), 20/27 (74%), 9/27 (33.3%), 12/27 (44.4%) and 5/29 (17.2%) specimens, respectively. VEGFR- 2 mRNA expression was found in 6/8 tumors extracted at first operation in patients with recurring tumors and in none of the control cases (p = 0.007). Microvessel density (MVD) and Ki-67 index values were generally higher in meningiomas with expression of angiogenic factors. The association of high Ki-67 index values with VEGF-mRNA expression was significant (p = 0.04). Time to recurrence was shorter in patients with high MVD than in patients with low MVD (p = 0.027). High MVD correlates with unfavorable prognosis in our series of recurring meningioma. VEGF and its receptors are frequently expressed in meningiomas and seem important for tumor growth and recurrence. Thus, anti-VEGF therapy in aggressive meningioma seems rational from a pathobiological point of view.