Low Microvessel Density Research Articles

Symptomatic carotid atherosclerotic disease: correlations between plaque composition and ipsilateral stroke risk.

For symptomatic patients with carotid artery stenosis, the risk benefit for surgical intervention may vary among patient groups. Various modalities of plaque imaging have been promoted as potential tools for additional risk stratification, particularly in patients with moderate stenosis. However, it remains uncertain to what extent carotid plaque components predict risk of future ipsilateral ischemic stroke. In 2 large atherosclerotic carotid plaque biobank studies, we related histological characteristics of 1640 carotid plaques with a validated risk model for the prediction of individual 1- and 5-year stroke risk. No significant heterogeneity between the studies was found. Predicted 5-year stroke risk (top versus bottom quartile) was related to plaque thrombus (odds ratio, 1.42; 95% confidence interval, 1.11-1.89; P=0.02), fibrous content (0.65; 0.49-0.87; P=0.004), macrophage infiltration (1.41; 1.05-1.90; P=0.02), high microvessel density (1.49; 1.05-2.11; P=0.03), and overall plaque instability (1.40; 1.05-1.87; P=0.02). This association was not observed for cap thickness, calcification, intraplaque hemorrhage, or lymphocyte infiltration. Plaques removed within 30 days of most recent symptomatic event were most strongly correlated with predicted stroke risk. Features of the vulnerable carotid plaque, including plaque thrombus, low fibrous content, macrophage infiltration, and microvessel density, correlate with predicted stroke risk. This study provides a basis for plaque imaging studies focused on stroke risk stratification.

The Morphological Evaluation of Walker 256 Tumors after Antiangiogenetic Therapy in Rats

Abstract Sunitinib Malate is a multi-targeted receptor tyrosine kinase inhibitor, including the vascular endothelial growth factor receptors, and is used for treatment of pancreatic neuroendocrine, renal cell carcinoma and gastrointestinal stromal tumors. The formation of new blood vessels (angiogenesis) may consequently lead to the development of metastases and so the aim of this study was to evaluate the microvessel density (MVD) after sunitinib malate treatment. In this study 11 albino Wistar rats were inoculated subcutaneously with Walker 256 tumor (breast carcinoma cells) and after 5 weeks, 5 rats were administered sunitinib malate at a dosage of 40 mg/kg every 3 days for 2 weeks. After another 4 weeks the tumors were removed and morphologically evaluated (weighted, measured). The MVD determined and quantified by calculating the average between 3 random fields. In the tumoral stroma we calculated also the vascular area. The immunohistochemical analysis of the neovascularization was performed using an anti-rat antibody against CD34. The difference between tumor weights were statistical significant (p 0.05) (32.55 ± 18.83; 18.6 ± 4.734).  As a conclusion, the rats treated with Sunitinib malate did not show a lower microvessel density comparative to the untreated ones. The number of rats used in this research was too small and further studies are needed.

Expression of GRIM-19 in missed abortion and possible pathogenesis

To study the expression of a gene associated with retinoid-interferon (IFN)-induced mortality 19 (GRIM-19) in the villi of patients with missed abortion and possible pathogenesis. Case-control study. University hospital. Villous samples were collected from a total of 50 women with missed abortion and 50 women with normal pregnancies. Villous samples from the missed-abortion group were collected by curettage or manual vacuum aspiration. The control villous samples were obtained by vacuum aspiration. Protein and messenger RNA levels of GRIM-19 in villous samples were measured by Western blot and real-time polymerase chain reaction, and the location of GRIM-19 in trophoblasts was detected by immunohistochemistry. Apoptotic cells and microvessel density in villous sample were assayed by TUNEL and immunostaining. Mitochondrial membrane potential and apoptotic changes of the HTR-8/SVneo cell line in response to down-regulation of GRIM-19 were evaluated by 5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide and annexin V/propidium iodide. Vascular endothelial growth factor production was measured by ELISA. GRIM-19 protein and messenger RNA levels in the villi from women with missed abortion were found to be significantly lower than in women who had normal pregnancies. Many more apoptotic cells and lower microvessel density were detected in villifrom patients with missed abortion. Knockdown of GRIM-19 in HTR-8/SVneo cells gave rise to a collapse of mitochondrial membrane potential and increase in apoptosis. Simultaneously, the vascular endothelial growth factor secretion in the HTR-8/SVneo cells culture medium decreased as GRIM-19 became down-regulated. The GRIM-19 deficiency in the villus may be associated with missed abortion via increasing apoptosis and affecting angiogenesis.

Microvascular effects of the low molecular weight heparins in a colorectal xenograft model: an intravital microscopy study

BackgroundLow molecular weight heparins (LMWHs) are the cornerstone of the prevention of thromboembolic events in surgical patients. Recent clinical data suggest that LMWHs might improve survival of cancer patients, independent of their anticoagulant effect. The anti-cancer mechanism of LMWHs is incompletely understood, but may include effects on tumor angiogenesis. We assessed the effects of LMWHs on tumor angiogenesis and microcirculation in a mouse colorectal xenograft model using in vivo microscopy in window chambers. MethodsHT29 human colorectal cancers were implanted in dorsal skinfold window chambers in athymic mice. Animals (n = 8 per group) were treated with 200 IU of nadroparin, enoxaparin, or saline for 8 d. Repeated intravital fluorescence microscopy was performed to calculate functional microcirculatory parameters as follows: number (N) and length (L) of microvessels, vascular area fraction (AF), and red blood cell velocity. Microvessel density, microvessel fractal dimension, and pericyte coverage were assessed histologically. ResultsActive angiogenesis was observed in control animals, resulting in a significant increase in N, L, and AF. In nadroparin- and enoxaparin-treated animals, however, AF did not change significantly over time and N and L remained significantly lower than untreated animals on day 7. Compared with control animals, nadroparin- and enoxaparin-treated animals showed a significantly lower microvessel density, but a higher pericyte coverage index, indicating a more mature microvessel network. ConclusionsThe LMWHs nadroparin and enoxaparin inhibit tumor angiogenesis and result in microvessel normalization in this in vivo observed colorectal xenograft model.

Abstract 2691: Smo-targeted sonic hedgehog signaling inhibitor enhances therapeutic antibody deposition and efficacy in a patient-derived pancreatic ductal adenocarcinoma model

Abstract Tumor stroma supports growth, invasion, and treatment resistance of pancreatic ductal adenocarcinoma (PDAC), and in conjunction with hypovascularity, constitutes a barrier that minimizes tumor cell drug exposure. Targeting tumor stroma is an attractive strategy in PDAC, and Smo-binding inhibitors are reported to mediate stromal thinning and increased microvessel density in PDAC models. No information exists as to the effect of sHHI upon deposition of macromolecular therapeutic agents such as monoclonal antibodies (mAb). Therefore we investigated effects of sHHI LDE225 upon tumor vascularity, mAb deposition, and mAb therapeutic efficacy in a patient-derived xenograft (PDX) model of PDAC in SCID mice. PDAC tumor #18269 was established in SCID mice and has retained abundant stroma and low microvessel density reminiscent of the original patient tumor. It expresses EGF receptor. Tumor-bearing mice were dosed orally with LDE225 (40 mg/kg) for 10 days, and pharmacodynamic biomarkers of sHHI effect were quantified. Mouse-specific TaqMan probes demonstrated high Gli1 expression in stromal cells, which was deeply suppressed within 3 days of sHHI dosing. Human-specific probes showed little Gli1 expression in tumor cells and no effect of sHHI treatment. Over 10 days of sHHI treatment, tumor microvessel density did not change significantly (based on CD31 staining), but the fraction of functional vessels increased significantly by day 7 (based on staining with fluorescent Lycopersicon esculentum lectin injected IV prior to tumor harvest). Fluorescently labeled mAbs that were tumor-targeted (cetuximab; anti-EGFR) or non-targeted (anti-topotecan) were co-injected on d7 of sHHI treatment. Compared to untreated controls, tumor exposure (AUC) of cetuximab more than doubled, and was observed to distribute to regions of the tumor enriched in tumor cells. Deposition of non-targeted mAb also was increased relative to controls, but distributed more to stromal areas than tumor cell areas. To investigate whether the enhanced deposition of cetuximab mediated by sHHI pretreatment would enhance efficacy, cetuximab was administered on d 5, 8, 11 of a 10-day sHHI or vehicle (control) regimen. This sequence was repeated 6 times, with an inter-cycle gap of 5 days. Rituximab (anti-human CD20) was the non-targeted control. Cetuximab alone showed no therapeutic benefit. However, cetuximab combined with sHHI suppressed tumor progression for more than 80 days, and median time to study endpoint was more than 250% longer than for all other treatment groups except sHHI-alone, which mediated a 30% increase. Conclusions: increased perfusion and vascular permeability mediated by sHHI pretreatment permits access of therapeutic mAb to otherwise inaccessible regions of the tumor interior, and reveals anti-tumor activity of a mAb that previously failed clinically for PDAC treatment. Citation Format: Jun Wang, Yang Qu, Rose Pitoniak, Sheryl A. Trueman-Fatallah, Bonnie L. Hylander, Elizabeth A. Repasky, Wen Wee Ma, Robert M. Straubinger. Smo-targeted sonic hedgehog signaling inhibitor enhances therapeutic antibody deposition and efficacy in a patient-derived pancreatic ductal adenocarcinoma model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2691. doi:10.1158/1538-7445.AM2014-2691

Transfection of wtp53 and Rb94 Genes Into Retinoblastomas of Nude Mice by Ultrasound-Targeted Microbubble Destruction

Using ultrasound-targeted microbubble destruction (UTMD), we transfected both wild-type p53 (wtp53) and Rb94 genes into retinoblastomas (RBs) of nude mice to investigate the inhibitory role of these two genes in RB development. The 40 tumor-bearing mice, which had been established by sub-retinal injection of an HXO-Rb44 cell suspension, were randomly divided into five groups: blank control group (C); blank plasmid group (M); wtp53 plasmid group (p53); Rb94 plasmid group (Rb94); wtp53 + Rb94 plasmid group (p53 + Rb94). For preparation of the DNA-loaded microbubbles, a pre-determined amount of blank plasmid, pVIVO1-p53, pVIVO1-Rb94 or pVIVO1-p53-Rb94 was added and mixed with the microbubbles. Then, these DNA-loaded microbubbles were respectively transfected into the animal model by UTMD. Vascular endothelial growth factor level and microvessel density of the tumor were determined by immunohistochemical staining. Apoptosis of tissues was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Expression of wtp53 and Rb94 at both the gene and protein levels was detected by RT-PCR (reverse transcription polymerase chain reaction) and Western blot, respectively. Transfection of both genes had greater inhibitory effects on RB development and resulted in lower levels of vascular endothelial growth factor, lower microvessel density and more obvious apoptosis than single-gene transfection (p < 0.05). The results indicate that the transfection of both genes into the RB by UTMD more strongly inhibited RB growth than transfection of a single gene.

Cerebral small-resistance artery structure and cerebral blood flow in normotensive subjects and hypertensive patients.

The aim of this study was to prospectively investigate whether the structure of cerebral small-resistance arteries is related to cerebral perfusion parameters as measured with dynamic susceptibility-weighted contrast magnetic resonance imaging (DSC-MRI) in a selected cohort of hypertensive and normotensive patients. Ten hypertensive and 10 normotensive patients were included in the study. All patients underwent neurosurgical intervention for an intracranial tumor and were investigated with DSC-MRI at 1.5 T. Cerebral small-resistance arteries were dissected from a small portion of morphologically normal cerebral tissue and mounted on an isometric myograph for the measurement of the media-to-lumen (M/L) ratio. A quantitative assessment of cerebral blood flow (CBF) and volume (CBV) was performed with a region-of-interest approach. Correlation coefficients were calculated for normally distributed variables. The institutional review board approved the study, and informed consent was obtained from all patients. Compared with normotensive subjects, hypertensive patients had significantly lower regional CBF (mL/100 g/min) in the cortical grey matter (55.63 ± 1.90 vs 58.37 ± 2.19, p < 0.05), basal ganglia (53.34 ± 4.39 vs 58.22. ± 4.33, p < 0.05), thalami (50.65 ± 3.23 vs 57.56 ± 4.45, p < 0.01), subcortical white matter (19.32 ± 2.54 vs 22.24 ± 1.9, p < 0.05), greater M/L ratio (0.099 ± 0.013 vs 0.085 ± 0.012, p < 0.05), and lower microvessel density (1.66 ± 0.67 vs 2.52 ± 1.28, p < 0.05). A statistically significant negative correlation was observed between M/L ratio of cerebral arteries and CBF in the cortical grey matter (r = -0.516, p < 0.05), basal ganglia (r = -0.521, p < 0.05), thalami (r = -0.527 p < 0.05), and subcortical white matter (r = -0.612, p < 0.01). Our results indicate that microvascular structure might play a role in controlling CBF, with possible clinical consequences.

Does expression of glucose transporter protein-1 relate to prognosis and angiogenesis in osteosarcoma?

The survival of patients who present with nonmetastatic extremity osteosarcoma has dramatically improved, but there are some patients who do not respond to chemotherapy. The ability to identify patients with a poorer prognosis might allow us to target different therapy for these patients. Glucose transporter protein-1 (Glut-1), one of the key factors in glucose metabolism, has been reported to be an independent prognostic factor in various tumors. However, little is known about the role of the Glut-1 pathway in osteosarcoma. We asked (1) if Glut-1 expression is a prognostic marker for survival in patients with osteosarcoma, and (2) if there is a relationship between Glut-1 expression and tumor angiogenesis. Thirty-seven patients with resectable high-grade osteosarcomas treated between 1982 and 2007 were reviewed retrospectively. Patients were excluded if representative biopsy material and followup data were not available. The expression of Glut-1 and the number of CD34-positive microvessels for angiogenic activity were measured immunohistochemically. The median followup was 6 years 6 months (range, 11-211 months). Survival analyses were evaluated using the Kaplan-Meier method and the Cox proportional hazards model. The association between Glut-1 expression and microvessel density was analyzed using Student's t-test and chi-square test. For 12 (32.4%) of 37 patients with osteosarcoma, the expression of Glut-1 was positive, with four patients (10.8%) showing strong expression of Glut-1 protein. The expression of Glut-1 correlated with a shorter disease-free survival period (relative risk, 20.13; 95% CI, 1.77-229.3; p=0.0016). The microvessel density mean value of positive Glut-1 expression (mean±SD, 26.5±19.4) was lower than that of negative expression (mean±SD, 46.4±35.3; Student's t-test, p=0.038). When more than 50 was defined as a high microvessel density, positive expression of Glut-1 was significantly associated with low microvessel density (chi-square test, p=0.049). These findings indicate that Glut-1 is a potential predictor of survival in patients with osteosarcoma and that glucose metabolism may be negatively associated with angiogenesis. If substantiated in larger numbers of patients, these findings might stimulate the development of novel treatments for patients with a poorer prognosis. Level III, prognostic study. See the Instructions for Authors for a complete description of levels of evidence.

P16.23 * A COMPARISON OF 18F-FET AND 18F-FLT MICROPET FOR THE ASSESSMENT OF ANTI-VEGF EFFICACY IN AN ORTHOTOPIC MURINE MODEL OF GLIOBLASTOMA

BACKGROUND: The radiolabeled amino acid O-(2-18F-fluoroethyl)-L-tyrosine (18F-FET) and thymidine analog 3'-deoxy-3'-18F-fluorothimidine (18F-FLT) are widely used for positron emission tomography (PET) brain tumor imaging; however, results from comparative studies are scarce. Due to the limited transport of FLT across the intact blood-brain barrier (BBB) we hypothesize that anti-VEGF response assessment with FLT PET primarily reflects changes in the BBB, whereas FET PET is less affected by the tumor vasculature. The aim of this study was therefore to evaluate and compare the value of FLT and FET MicroPET for anti-VEGF response assessment in orthotopic human glioma xenografts. METHODS: Cells of human glioblasoma multiforme (GBM) neurosphere culture (NGBM_CPH048p6_LUC) were injected orthotopically in NMRI nude mice. At confirmed tumor take, mice were treated with anti-VEGF therapy (B20-4.1) or saline as control. Treatment response was followed by weekly bioluminescence, 18F-FLT and 18F-FET MicroPET/CT. The end-point was survival and brains from sacrificed mice were used for immunohistochemistry and subsequent quantification of the Ki67 proliferation index and micro-vessel density (MVD). RESULTS: The relative 18F-FET tumor-to-brain of (T/B) ratio of SUVmax was significantly decreased after one week (99 ± 6%, n = 6 vs. 124 ± 9%, n = 5; p = 0.04) and after two weeks (106 ± 10%, n = 5 vs. 156 ± 17%, n = 4; p = 0.03) in the B20-4.1 group as compared with the control group. However, with 18F-FLT MicroPET there was no significant difference in the T/B ratio of SUVmax neither after one week of treatment (134 ± 12%, n = 6 vs. 144 ± 6%, n = 6; p = 0.49) nor after two weeks of treatment (174 ± 11%, n = 5 vs. 208 ± 30%, n = 5; p = 0.34). We found a significant lower MVD in the B20-4.1 group as compared to the control group (66 ± 8%, n = 8 vs. 100 ± 10%, n = 7; p = 0.02). However, we found no difference in the Ki67 proliferation index in the treatment group as compared to the control group (18.9 ± 1%, n = 7 vs. 19.8 ± 2%; n = 6; p = 0.7). In addition, the median survival was not prolonged in the treatment group as compared to the control group (19 vs. 21 days, p = 0.34). CONCLUSION: In NGBM_CPH048p6_LUC xenografts B20-4.1 anti-VEGF treatment induced changes in the MVD and in 18F-FET uptake without affecting the Ki67 proliferation index. Surprisingly and in contradiction with the hypothesis, the 18F-FLT uptake was not affected by the changes in the MVD.

BCL-2, topoisomerase IIα, microvessel density and prognosis of early advanced breast cancer patients after adjuvant anthracycline-based chemotherapy.

Purpose The aim of this retrospective study was to investigate the effect of B cell lymphoma 2 (BCL-2) expression on disease-free survival (DFS) in 172 early breast cancer (BC) patients treated with anthracycline-based adjuvant chemotherapy. We have reanalysed follow-up data in these patient groups, and therefore, the relation between DFS and other tumour biological features [expression of oestrogen (ER) and progesterone (PgR) receptors, cytokeratin 5/6 (CK5/6), HER2, topoisomerase IIα (TOPOIIα), Ki-67, P53 and microvessel density (MVD)] studied previously (Biesaga et al. in Breast 20(4):338–350, 2011, doi:10.1016/j.breast.2011.03.002, Pathol Oncol Res 18(4): 949–960, 2012, doi:10.1007/s12253-012-9525-9) was also investigated.MethodTumour biological features were assessed immunohistochemically on paraffin-embedded sections obtained before treatment from 172 women with BC in stage T1–T2, N1–N2, M0.ResultsIn univariate analysis, longer DFS was found for patients having tumours with BCL-2 positivity (P = 0.005), low grade (P = 0.001), ER (P = 0.017) and PgR (P = 0.045) positivity, CK5/6 negativity (P = 0.021), low TOPOIIα expression (P = 0.003) and high MVD (P = 0.000). In multivariate analysis, BCL-2, TOPOIIα and MVD were independent parameters indicating patient prognosis. All patients (n = 18) characterized by tumour BCL-2 positivity, low TOPOIIα expression and high MVD survived 80 months without any evidence of cancer disease, whereas DFS for all other patients was significantly (P = 0.022) lower (76.5 %).ConclusionCombination of three parameters: BCL-2 positivity, low topoisomerase IIα expression and high MVD, allows to identify subgroup of BC patients with very good prognosis after adjuvant anthracycline-based chemotherapy.

Radiation-induced endothelial cell loss and reduction of the relative magnitude of the blood flow in the rat spinal cord

The main purpose of the present study was to examine the time-dependent alterations in the endothelial cell density that occur in the first 180 days after irradiation of the spinal cord and the functional role of these alterations in the spinal cord blood flow. An irradiated cervical spinal cord rat model (C2-T2 segment) was generated using a 60Co irradiator to deliver 30Gy. A significant loss of forelimb motor function was observed 180 days post-irradiation. The number of neurons in the anterior horn of the spinal cord began to decrease significantly 3 days post-irradiation compared with normal controls, reaching the lowest number at 90 days post-irradiation. A significant reduction in the endothelial cell density was observed from 14 days post-irradiation in the white matter and from 3 days post-irradiation in the gray matter. The lowest endothelial cell density was reached at 30 days post-irradiation in the white matter and at 60 days post-irradiation in the gray matter. A significant reduction in the microvessel density was observed from 3 days post-irradiation in both the white matter and the gray matter. The lowest microvessel density was reached at 90 days post-irradiation in both the white matter and the gray matter. A significant reduction in the relative magnitude of spinal cord blood flow was observed from 21 days post-irradiation. The lowest relative magnitude of spinal cord blood flow was reached at 90 days post-irradiation. We did not find any evidence of demyelination. The results revealed that a single 30-Gy irradiation dose resulted in impaired forelimb motor function, a decreased number of neurons, and reduced endothelial cell density, microvessel density and relative magnitude of spinal cord blood flow. However, a 30-Gy single-dose irradiation was not sufficient to induce demyelination in the rat spinal cord.

The combination of HTATIP2 expression and microvessel density predicts converse survival of hepatocellular carcinoma with or without sorafenib.

Our previous studies have demonstrated that sorafenib can promote the dissemination of hepatocellular carcinoma (HCC) through downregulation of HTATIP2, a suppressor of tumor growth and metastasis that is associated with inhibition of angiogenesis. Here, we investigated the predictive values of the HTATIP2 level and microvessel density (MVD) with or without sorafenib administration for HCC. Three independent cohorts were included. Using tissue microarray, we assessed the relationship between HTATIP2 expression/MVD and overall survival. The results showed that high HTATIP2 expression and a low MVD value were independent protective prognostic factors after curative HCC resection (297 cases/cohort 1); however, both parameters were converted to independent negative prognostic indicators for patients with postsurgical sorafenib treatment (69/143 cases/cohort 2; P<0.05 for all). This same relationship was observed in patients that received sorafenib treatment for advanced HCC (83 cases/cohort 3; efficacy measures and survival analyses, P<0.05 for all). Moreover, the combination of HTATIP2 and MVD had better power to predict patient death and disease recurrence (P<0.001 for both). We conclude that the combination of HTATIP2 and MVD predicts the converse survival of HCC with or without sorafenib intervention. Our findings can assist in the selection of candidates for personalized treatment with sorafenib.

The expression of beclin-1, an autophagic gene, in hepatocellular carcinoma associated with clinical pathological and prognostic significance.

BackgroundA role for autophagy, a conserved cellular response to stress, has recently been demonstrated in human cancers. Aberrant expression of Beclin-1, an important autophagic gene, has been reported in various human cancers. In the present study, we investigated the significance and relationship between Beclin-1 expression and cell proliferation, apoptosis, microvessel density (MVD) and clinical pathological changes or prognosis in human hepatocellular carcinoma (HCC).MethodsA total of 103 primary HCC patients were involved in the study. Expression of Beclin-1, PCNA, NET-1, Bcl-2, Bax, Survivin in cancer cells and CD34 in stromal microvessels were evaluated immunohistochemically in tissue microarrays comprising 103 cases of HCC and 57 matched adjacent nontumor liver tissues. Correlations between clinicopathological characteristics and survival of HCC patients were explored.ResultsThe positive rate of Beclin-1 was significantly lower in HCC tissues than adjacent tissues (72.8 vs. 89.5%, χ2 = 6.085, P = 0.015). In HCC, Beclin-1 expression was negatively correlated with cirrhosis background (r = −0.216, P = 0.029), Edmondson grade (r = −0.249, P = 0.011), vascular invasion (r = −0.246, P = 0.012), PCNA (r = −0.242, P = 0.014), NET-1 (r = −0.245, P = 0.013), anti-apoptosis protein Bcl-2 (r = −0.245, P = 0.013) and MVD (r = −0.292, P = 0.003), and positively correlated with pro-apoptosis protein Bax (r = 0.242, P = 0.014).Significant differences in the 5-year survival rates were seen among patients with Beclin-1 strong positive (++) (59.1%, 13/22), moderate positive (+) (28.3%, 15/53) and weak negative expression (−) (14.6%, 7/28) (P = 0.043). Significant differences were detected between Beclin-1 (++) and either Beclin-1 (+) (P = 0.036) or Beclin-1 (−) groups (P = 0.008), but no significant difference between Beclin-1 (+) and Beclin-1 (−) groups (P = 0.281) was observed.Survival rates were positively related to high Beclin-1 co-expressed with low PCNA, NET-1, or Bcl-2, lower MVD, and high Bax. Univariate and multivariate Cox regression analysis revealed that Beclin-1 expression was an independent indicator for overall survival in HCC patients (P < 0.05).ConclusionsThe pathogenesis and progression of HCC are associated with reduced autophagy. The expression of Beclin-1 and Bax in HCC tissues may provide a synergistic effect towards inhibiting HCC proliferation, infiltration, metastasis and angiogenesis. Beclin-1 expression may be a valuable prognostic marker of HCC.

Immuno-Expression of Endoglin and Smooth Muscle Actin in the Vessels of Brain Metastases. Is There a Rational for Anti-Angiogenic Therapy?

Despite ongoing clinical trials, the efficacy of anti-angiogenic drugs for the treatment of brain metastases (BM) is still questionable. The lower response rate to anti-angiogenic therapy in the presence of BM than in metastatic disease involving other sites suggests that BM may be insensitive to these drugs, although the biological reasons underlining this phenomenon are still to be clarified. With the aim of assessing whether the targets of anti-angiogenic therapies are actually present in BM, in the present study, we analyzed the microvessel density (MVD), a measure of neo-angiogenesis, and the vascular phenotype (mature vs. immature) in the tumor tissue of a series of BM derived from different primary tumors. By using immunohistochemistry against endoglin, a specific marker for newly formed vessels, we found that neo-angiogenesis widely varies in BM depending on the site of the primary tumor, as well as on its histotype. According to our results, BM from lung cancer displayed the highest MVD counts, while those from renal carcinoma had the lowest. Then, among BM from lung cancer, those from large cell and adenocarcinoma histotypes had significantly higher MVD counts than those originating from squamous cell carcinoma (p = 0.0043; p = 0.0063). Of note, MVD counts were inversely correlated with the maturation index of the endoglin-stained vessels, reflected by the coverage of smooth muscle actin (SMA) positive pericytes (r = −0.693; p < 0.0001). Accordingly, all the endoglin-positive vessels in BM from pulmonary squamous cell carcinoma and renal carcinoma, displayed a mature phenotype, while vessels with an immature phenotype were found in highly vascularized BM from pulmonary large cell and adenocarcinoma. The low MVD and mature phenotype observed in BM from some primary tumors may account for their low sensitivity to anti-angiogenic therapies. Although our findings need to be validated in correlative studies with a clinical response, this should be taken into account in therapeutic protocols in order to avoid the adverse effects of useless therapies.

MicroRNA-26a suppresses angiogenesis in human hepatocellular carcinoma by targeting hepatocyte growth factor-cMet pathway

MicroRNA (miR)-26a can suppress tumor growth and metastasis of hepatocellular carcinoma (HCC). Since angiogenesis is important for tumor growth and metastasis, we investigated the possible roles of miR-26a in tumor angiogenesis. Down-regulation of miR-26a was found to correlate with an increased angiogenic potential of HCC. Through gain- and loss-of-function studies, miR-26a was demonstrated to significantly inhibit vascular endothelial growth factor A (VEGFA) expression in HCC cells and then suppress the promoting effects of HCC cells on in vitro proliferation, migration, and capillary tube formation of endothelial cells, as well as in vivo tumor angiogenesis of HCC. Hepatocyte growth factor (HGF) was identified as a target of miR-26a. HGF simulation antagonized the effects induced by miR-26a up-regulation. In contrast, silencing HGF induced similar effects to miR-26a. We further found that miR-26a exerted its antiangiogenesis function, at least in part, by inhibiting HGF-hepatocyte growth factor receptor (cMet) and its downstream signaling pathway, in turn, suppressing VEGFA production in HCC cells and impairing VEGFR2-signaling in endothelial cells. HCC patients who had high miR-26a, low HGF, low VEGFA, or low microvessel density (MVD) in tumor tissues had a better prognosis with longer overall survival (OS) and time to recurrence (TTR). In multivariate analysis, miR-26a, or in combination with HGF, was demonstrated to be an independent prognostic indicator for OS and TTR of HCC patients. miR-26a could suppress tumor angiogenesis of HCC through HGF-cMet signaling, and it is a new hopeful therapeutic target and prognostic marker for HCC.

The clinical implication of cancer-associated microvasculature and fibroblast in advanced colorectal cancer patients with synchronous or metachronous metastases.

BackgroundWe aimed to evaluate the clinical significance of microvessel density (MVD), lymphatic vessel density (LVD), and cancer-associated fibroblasts (CAFs) in relation to tumor location in advanced colorectal cancer (CRC).MethodsUsing immunohistochemistry, we examined 181 advanced CRC patients for CD31 and D2-40 to measure MVD and LVD, respectively, α-smooth muscle actin (SMA) and desmin to identify CAFs, and PTEN to examine genetic changes of CAFs. To evaluate the regional heterogeneity of these properties, we examined tissue from four sites (the center and periphery of the primary cancer, a distant metastasis, and a lymph node metastasis) in each patient.ResultsMVD, LVD, and CAFs showed significant heterogeneity with respect to the tumor location. LVD was the greatest in the center of the primary cancers and the amount of CAFs was the lowest in distant metastases. In distant metastases, those from the lung had higher LVD and MVD, but fewer CAFs than those from the liver, peritoneum, or ovary. Patients with low MVD and LVD in the center of the primary cancer had worse outcomes and patients with few CAFs in distant metastases and in the primary tumor had a lower survival rate. PTEN expression in CAFs in distant metastases was lost in 11 of 181 CRC patients (6.1%), which was associated with a worse prognosis.ConclusionsThe microenvironment, including cancer-associated microvasculature and fibroblasts, is heterogeneous with respect to the tumor location in CRC patients. Therefore, heterogeneity of microenvironments should be taken into account when managing CRC patients.

CD163 Expression Was Associated with Angiogenesis and Shortened Survival in Patients with Uniformly Treated Classical Hodgkin Lymphoma

BackgroundRecent studies have reported the prognostic value of tissue-associated magrophages (TAMs) in classical Hodgkin lymphoma (cHL). In addition, TAMs are implicated in the tumor angiogenesis. In this study, we examined the prognostic relevance of TAMs in relation to vascular endothelial growth factor (VEGF) expression and angiogenesis in uniformly treated cases of cHL.MethodsDiagnostic tissue from 116 patients with ABVD-treated cHL was evaluated retrospectively by immunohistochemical analysis for CD68, CD163 and VEGF expression and for CD31 expression as a measure of microvessel density (MVD).ResultsHigh CD163 expression (≥35% of cellularity) correlated with VEGF expression (Pearson’s Chi-square test, P = 0.008) and MVD (Spearman correlation coefficient 0.310, P<0.001). High CD163 expression was associated with inferior event-free survival (EFS, P = 0.005) and overall survival (OS, P<0.001) in univariate analysis. In multivariate analysis, high CD163 expression was strongly associated with inferior EFS (P = 0.043) and OS (P = 0.008). Patients with high MVD had a lower OS than those with low MVD, but the difference was not significant (P = 0.071, respectively). While high expression of CD68 was also associated with inferior EFS (P = 0.007), it showed no correlation with VEGF or MVD.ConclusionsOur data confirms that CD163 expression provides independent prognostic information in cHL. The correlation of CD163 with VEGF expression and MVD suggests the role of CD163-positive cells in tumor angiogenesis of cHL.

Expression of AEG-1 and microvessel density correlates with metastasis and prognosis of oral squamous cell carcinoma

Astrocyte elevated gene 1 (AEG-1) expression is up-regulated in various human cancers and plays an important role in tumorigenesis and progression. The aim of this study was to explore AEG-1 expression in oral squamous cell carcinoma (OSCC) and to assess whether it is associated with microvessel density (MVD), metastasis, and survival. Specimens from 87 patients with OSCC were investigated by immunohistochemistry staining for AEG-1 and MVD. By statistical analysis, we studied the correlations between the expression of AEG-1 and MVD and various clinicopathological factors, including overall survival (OS). We found that AEG-1 was highly expressed in 51.72% of OSCC. Expression was closely correlated with differentiation, clinical stage, T classification, and lymph node metastasis. The MVD had similar results. Expression of AEG-1 correlated positively with MVD. The lymph node metastatic rate in patients with high AEG-1/high MVD was significantly higher than in patients with high AEG-1/low MVD, low AEG-1/high MVD, or low AEG-1/low MVD. Patients with high AEG-1 expression showed far lower OS rates than those with low expression. For MVD, there were similar results. Only AEG-1 and MVD expression were independent prognostic factors for OS by multivariate analysis. Expression of AEG-1 may be correlated with tumor angiogenesis and metastasis and is a valuable prognostic factor in patients with OSCC.

Caractéristiques histologiques de la membrane induite selon son site d’implantation : sous-cutané, intra-musculaire ou au niveau de la perte de substance osseuse

Background The induced membrane technique was proposed as a treatment of large segmental bone defects. The influence of the surrounding tissues on its characteristics remains unknown. It is therefore not known which kind of plastic surgery procedure (muscular or facio-cutaneous flap) would optimize bone osteointegration within a bone defect reconstructed using the induced-membrane technique. Hypothesis We hypothesized that membrane characteristics could be influenced by the soft-tissue environment either subcutaneous or muscular. Objective To evaluate the histological characteristics of poly-methylmethacrylate (PMMA)-induced membranes in intramuscular, subcutaneous and bony environment (radius defects) at 2 steps: 1) spacer implantation; 2) secondary bone graft and its subsequent osteintegration after spacer removal. Methods PMMA-induced membranes were obtained in the three sites of 15 rabbits. Subsequent new bone formation was studied in the same environments in 24 other rabbits. Six weeks after the initial implantation, PMMA spacers were replaced with iliac autografs. Animals were euthanized at 2, 4, and 8 weeks postoperatively. Tissue samples were harvested and stained with hematoxylin and eosin. The histological characteristics of the membrane (thickness and microvessel density) and the newly-formed bone (cortical thickness) were quantitatively analyzed. Results The membranes in the subcutaneous sites developed quicker, were thicker and had the lowest microvessel density (P < 0.01). The membranes in the intramuscular sites developed later and were thinner (P < 0.01). The membranes in the osseous defects had the greatest microvessel density (P < 0.01). After bone grafting, induced membranes became thinner and their microvessel density decreased substantially, but maintained better in osseous site. The newly-formed bone that developed in the radius defects, had the thickest cortices (P < 0.01). Conclusions The evolution of membranes induced in the intramuscular and subcutaneous environments was close to that of the bone defect model, although bone formation appeared weaker. Level of evidence Basic science study III.

Bone marrow angiogenesis in patients presenting with differential Chinese medicine syndrome: Correlation with the clinico-pathological features of aplastic anemia

To explore differences in bone marrow angiogenesis seen in aplastic anemia (AA) patients presenting with differential Chinese medicine (CM) syndrome, and to correlate these differences with clinical pathology. Thirty-five patients were enrolled, including 18 with "yang deficiency syndrome" and 17 with "yin deficiency syndrome." Bone marrow biopsies and serum were collected. Microvessel density (MVD) and positive expression of vascular endothelial-derived growth factor (VEGF) were detected by immunohistochemisty. Hypoxia inducible factor -1α (HIF-1α), and VEGF expression were assayed by enzyme-linked immunoabsorbent assay (ELISA), serum lactate dehydrogenase (LDH) was tested by enzyme method and liquid chip technology was used to detected the expression of interleukin (IL)-2, IL-4, IL-6, IL-10, interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Counts for leukocytes, absolute neutrophils and platelets in "yin deficiency syndrome" were lower than those found in "yang deficiency syndrome" (P<0.05). MVD and VEGF expression, and the positive rate of CD34 and VEGF in bone marrow were lower in AA, especially in "yin deficiency syndrome" (P<0.01 or P<0.05). "Yin deficiency syndrome" displayed decreased VEGF and LDH expression, and enhanced expression of HIF-1α as compared to "yang deficiency syndrome" (P<0.05). Levels of IL-4 and IL-6 were higher in AA (P<0.01), but IL-10 was decreased (P<0.05). High TNF-α expression was seen in "yang deficiency syndrome" and IFN-γ expression was decreased in "yin deficiency syndrome" as compared with normals (P <0.01 and P<0.05, respectively). AA patients have lower MVD than normals, especially in "yin deficiency syndrome." MVD might differentially correlate to disease severity, and could be dependent on bone marrow or serum VEGF expression and LDH. Additionally, IL-2, IL-10, IL-4 and IFN-γ were negatively associated while IL-6 and TNF-α were positively associated with MVD.