Angiogenesis Parameters Research Articles

Proliferation of endothelial cells in neoangiogenesis of human cortical glioblastoma

Abstract In 1982 Vilanova et al. quantitatively described the neovessel area in glioblastoma and suggested zonal differences in vessel surface area. In this study we investigated specific cell proliferation and angiogenic patterns in the vessel compartment of cortical glioblastoma. We used Ki67, CD34 and SMA double immunohistochemical staining to quantitate vascular patterns and cell specific proliferation and presented glioblastoma with several parameters of angiogenesis. Endothelial cell proliferation was higher in complex and bizzare neovessels than in the simple and sprouting glioblastoma neovessels. There was a higher frequency of sprouting simple vessels in close proximity to the palisade and a higher frequency of bizzare vessels in the microzone distant to the palisade. Quantitatively presented for the first time, the neovessel proliferation patterns support cortical glioblastoma compartmentalization. The data obtained are relevant to medical doctors using neoangiogenesis in diagnosis, prognosis and therapy of neoplasia. The results obtained in 15 patients call for further investigation of endothelial cell/pericyte relationships and glioblastoma compartmentalization.

Correlative study of the parameters of dynamic contrast-enhanced MRI and angiogenesis in breast lesions

目的 探讨乳腺良、恶性病变MR动态增强(DCE)参数与肿瘤血管生成的关系.方法 对51例乳腺良、恶性病变患者行前瞻性DCE-MR检查,计算动态增强参数:最大增强线性斜率(Smax)、增强峰值(PH)、峰值时间(Tpeak).所有病例手术病理标本行免疫组织化学染色,测定微血管密度(MVD)计数和血管内皮生长因子(VEGF)表达,分析各动态增强参数与乳腺癌MVD和VEGF表达的相关性.并比较29例乳腺癌与12例乳腺纤维腺瘤、10例乳腺病、10例癌旁正常组织MVD计数和VEGF表达的差别.结果 乳腺癌Smax(2.04±1.8;r=0.807,P＜0.01)、PH(678±260;r=0.697,P＜0.01)与MVD呈正相关,Tpeak(69±38)与MVD呈负相关(r=-0.425,P＜0.05).乳腺癌组MVD计数[(65.09±15.81)个/200倍视野]明显高于纤维腺瘤组、乳腺腺病组及癌旁正常组织组(P值分别为0.043、0.018、0.002).69%(20/29)乳腺癌VEGF表达阳性,也明显高于其余各组(P值分别为0.035、0.007、0.001).乳腺癌边缘区域的MVD(60.38±24.14)个/200倍视野,高于中央区域(37.64±16.52)个/200倍视野;(t=2.635,P=0.016).腋窝淋巴结转移组的MVD(73.23±23.02)个/200倍视野,高于无转移组(59.34±18.03)个/200倍视野,(t=2.303,P=0.031).结论 乳腺癌部分MR-DCE参数与MVD、VEGF相关,能较客观地反映乳腺癌血管生成状况,其MVD计数和VEGF表达明显高于乳腺良性病变及癌旁正常组织。

Prognostic Value of Measurements of Angiogenesis in Serous Carcinoma of the Ovary

The study objective was to determine whether tumor vascularity correlates with patient survival, to compare newer semiautomated methods of angiogenesis assessment to older methods, and to determine if advanced image analysis methods can offer useful patient outcome data in serous ovarian cancer. Using the specific endothelial marker CD34, microvessel determinations were quantified in 132 serous ovarian tumors by manual counting at final magnifications of x 200 and x 400 in the most highly vascular areas. Computer-assisted image analysis microvessel counts, endothelial area estimates, and minimum spanning tree (MST) analysis of capillary architecture, which involves assessment of intercapillary distances, were correlated with traditional manual techniques.Manual, semiautomated, and advanced image analysis methods were found to be highly reproducible and express strong correlation with one another. Univariate cyclooxygenase analysis revealed angiogenesis parameters to be highly significant predictors for overall survival (OS) and disease-free survival. Multivariate cyclooxygenase analysis revealed maximum MST (P = 0.009), length MST (P = 0.005), 1 nearest neighbor (P <or= 0.01) and mean microvessel density (x 400) (P = 0.0001) to be significant predictors for OS, and mean endothelial area (P <or= 0.01) and stage (P = 0.001) significant predictors for disease-free survival. Despite showing prognostic significance on univariate analysis, most clinicopathologic parameters did not retain independent significance on multivariate analysis. Microvessel determination is an independent prognostic indicator for survival in patients with serous ovarian carcinoma. Computer-assisted image analysis is a highly reproducible method of assessment capable of accurately evaluating tumor vascularity. Minimum spanning tree analysis of capillary architecture was found to be the strongest prognosticator for OS, suggesting this to be a promising marker.

Microvessel density in normal lymph nodes and lymphomas of dogs and their correlation with vascular endothelial growth factor expression

Microvessel density is a frequently used parameter of angiogenesis, which is a complex multistep process necessary for tumor progression. The aim of this study was to compare the microvessel density of normal lymph node biopsies with those diagnosed with lymphoma in dogs. Furthermore, we sought to determine if there was any correlation between microvessel density and vascular endothelial growth factor expression in canine lymphoma, representing a potential target for anti-angiogenic therapy. Combined immunohistochemistry (von Willebrand factor) and lectin histochemistry was used to highlight microvessels in 40 untreated canine lymphomas and 14 normal lymph nodes. To evaluate microvessel density, the number of profiles of blood vessels per unit area was calculated. Fifty image fields (a total area of 5.68 mm 2) were sampled for each specimen in a systematic random, way. We found a significant difference between the microvessel densities (MVD) of normal and neoplastic lymph nodes (177 ± 35 versus 241 ± 72 microvessel profiles/mm 2). Classifying lymphoma samples according to the working formulation and the Kiel classification system revealed no significant differences in MVD between different grade malignancies. Immunohistochemical demonstration of the proangiogenic protein vascular endothelial growth factor showed expression in 60% of canine lymphomas, although there was no correlation between microvessel density and vascular endothelial growth factor expression. As an increase in tumor angiogenesis was observed in lymphoma samples compared to normal canine lymph node tissue, additional anti-angiogenic therapy, besides conventional chemotherapy as a lymphoma treatment may be effective. The optimal target among many pro-angiogenic factors has yet to be elucidated.

High-grade clear cell renal cell carcinoma has a higher angiogenic activity than low-grade renal cell carcinoma based on histomorphological quantification and qRT–PCR mRNA expression profile

Clear cell renal cell carcinoma (CC-RCC) is a highly vascularised tumour and is therefore an attractive disease to study angiogenesis and to test novel angiogenesis inhibitors in early clinical development. Endothelial cell proliferation plays a pivotal role in the process of angiogenesis. The aim of this study was to compare angiogenesis parameters in low nuclear grade (n=87) vs high nuclear grade CC-RCC (n=63). A panel of antibodies was used for immunohistochemistry: CD34/Ki-67, carbonic anhydrase IX, hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF). Vessel density (MVD – microvessel density), endothelial cell proliferation fraction (ECP%) and tumour cell proliferation fraction (TCP%) were assessed. mRNA expression levels of angiogenesis stimulators and inhibitors were determined by quantitative RT–PCR. High-grade CC-RCC showed a higher ECP% (P=0.049), a higher TCP% (P=0.009), a higher VEGF protein expression (P<0.001), a lower MVD (P< 0.001) and a lower HIF-1α protein expression (P=0.002) than low-grade CC-RCC. Growth factor mRNA expression analyses revealed a higher expression of angiopoietin 2 in low-grade CC-RCC. Microvessel density and ECP% were inversely correlated (Rho=−0.26, P=0.001). Because of the imperfect association of nuclear grade and ECP% or MVD, CC-RCC was also grouped based on low/high MVD and ECP%. This analysis revealed a higher expression of vessel maturation and stabilisation factors (placental growth factor, PDGFB1, angiopoietin 1) in CC-RCC with high MVD, a group of CC-RCC highly enriched in low nuclear grade CC-RCC, with low ECP%. Our results suggest heterogeneity in angiogenic activity and vessel maturation of CC-RCC, to a large extent linked to nuclear grade, and, with probable therapeutic implications.

Integrin‐linked kinase is an essential mediator for T‐cadherin‐dependent signaling via Akt and GSK3β in endothelial cells

Glycosylphosphatidylinositol-anchored T-cadherin (T-cad) influences several parameters of angiogenesis including endothelial cell (EC) differentiation, migration, proliferation, and survival. This presupposes signal transduction networking via mediatory regulators and molecular adaptors since T-cad lacks transmembrane and cytosolic domains. Here, using pharmacological inhibition of PI3K, adenoviral-mediated T-cad-overexpression, siRNA-mediated T-cad-depletion, and agonistic antibody-mediated ligation, we demonstrate signaling by T-cad through PI3K-Akt-GSK3beta pathways in EC. T-cad-overexpressing EC exhibited increased levels and nuclear accumulation of active beta-catenin, which was transcriptionally active as shown by increased Lef/Tcf reporter activity and cyclin D1 levels. Cotransduction of EC with constitutively active GSK3beta (S9A-GSK3beta) abrogated the stimulatory effects of T-cad on active beta-catenin accumulation, proliferation, and survival. Integrin-linked kinase (ILK), a membrane proximal upstream regulator of Akt and GSK3beta, was considered a candidate signaling mediator for T-cad. T-cad was present in anti-ILK immunoprecipitates, and confocal microscopy revealed colocalization of T-cad and ILK within lamellipodia of migrating cells. ILK-siRNA abolished T-cad-dependent effects on (Ser-473)Akt/(Ser-9)GSK3beta phosphorylation, active beta-catenin accumulation, and survival. We conclude ILK is an essential mediator for T-cad signaling via Akt and GSK3beta in EC. This is the first demonstration that ILK can regulate inward signaling by GPI-anchored proteins. Furthermore, ILK-GSK3beta-dependent modulation of active beta-catenin levels by GPI-anchored T-cad represents a novel mechanism for controlling cellular beta-catenin activity.

Assessment of angiogenesis expression and its relationship with prognosis of colorectal cancer by conventional and computer-assisted histopathological image analysis

To quantify the degree of angiogenesis by conventional method (microvessel density, MVD) and computerized method (endothelial area, EA), and to evaluate their relationships with the prognosis of patients operated on for colorectal adenocarcinoma. Tumoral angiogenesis was studied by means of an immunohistochemical technique, using CD 34, on 126 patients; to quantify the angiogenesis, MVD (defined as number of microvessels per mm(2)) and EA measurement (defined as the area occupied by EA in the microscope field). A computerized method, IMAGELab software was utilized to quantify endothelial area. The mean number of microvessels was 128.6 MV/mm(2) (SD = 44.5) and the mean EA was 4.3% (SD = 2.1). The Pearson method demonstrated a low correlation coefficient between MVD and EA (r = 0.429). No relationship between MVD and EA was observed with regard to relapse-free interval and overall survival. The histological analysis of angiogenesis expression in patients with colorectal adenocarcinoma can be performed either by computer-assisted image analysis of endothelial area or by conventional microvessels counting. Both methods did not show any significant relationship between these angiogenesis parameters with relapse-free interval and overall survival.

Proliferating Endothelial Cells and Leukocyte Infiltration as Prognostic Markers in Colorectal Cancer

Leukocyte infiltration in tumors is dependent on angiogenic potential. In this study we aimed to retrospectively investigate the angiogenic potential in archival colorectal cancer (CRC) tissues and its relationship to amount and composition of the inflammatory infiltrate. In tumor tissues of 117 CRC patients with a 12-year follow-up, microvessel density (MVD) and proliferating endothelial cells (ECs) were assessed by CD31/CD34 double staining with the proliferation marker Ki-67. Leukocyte infiltration was determined by using CD45, CD3, CD8, CD16, CD20, and CD68 antibodies in peritumoral, tumor stroma, and intratumoral areas. Proliferating ECs, but not MVD, are correlated to Dukes' stage and survival in CRC (P < .05). This parameter correlated significantly with the expression of vascular endothelial growth factor (r = 0.82; P < .012). The number of inflammatory cells in the tumor stroma and cells infiltrated into the tumor cell nests, but not of peritumoral leukocytes, predicted patient survival. This was most obvious for T lymphocytes (CD3; P < .05) and polymorphonuclear cells (CD16; P < .04). We found a significant relationship between angiogenesis parameters and infiltrated leukocytes (r = -0.70; P < .02). Combination of high numbers of infiltrated leukocytes and low amounts of proliferating ECs demonstrated to be an improved prognostic value compared with either parameter alone (P < .006). We found a correlation between the intrinsic tumor parameters of ongoing angiogenesis and leukocyte infiltration with prognosis and survival in CRC. These findings have a potential impact on therapeutic applications for both antiangiogenesis as well as immunotherapy.

Prognostic value of bone marrow angiogenesis in multiple myeloma: Use of light microscopy as well as computerized image analyzer in the assessment of microvessel density and total vascular area in multiple myeloma and its correlation with various clinical, histological, and laboratory parameters

We studied the prognostic value of parameters of angiogenesis on bone marrow biopsies in newly diagnosed multiple myeloma (MM) patients. Angiogenesis parameters studied were the microvessel count done manually on light microscopy (MVD-A), microvessel count done by using computerized image analyzer (MVD-B), and total vascular area (TVA) measured by computerized image analyzer. One hundred ten newly diagnosed cases of MM treated at Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, were analyzed with respect to clinical features, laboratory findings, histological features, angiogenesis parameters, and responses to the treatment on follow-up. Twenty age- and sex-matched controls were studied for comparing with angiogenesis of the test cases. Bone marrow microvessels were examined using immunohistochemical staining for CD34. MVD-A (range 4.9-85.2; mean 28.2; SD 19.4), MVD-B (range 2.0-26.9; mean 11.7; SD 5.9), and TVA measured in percentage (range 0.1-17.1; mean 2.4; SD 2.5) were measured for test cases (n = 110). Grading of angiogenesis parameters of the test cases were done; such that angiogenesis parameters of controls (taken as baseline) were grade I. There was a statistically highly significant correlation between (MVD-A vs MVD-B, pcc = 0.92; MVD-A vs TVA, pcc = 0.78; MVD-B vs TVA, pcc = 0.76). The myeloma cases had significantly higher angiogenesis parameters when compared with controls (Kruskall-Wallis test, P < 0.001). "Complete responders" (n = 38/110) had significant lower angiogenesis (Mann-Whitney U test, P < 0.001) than "nonresponders" (n = 72/110). On treatment follow-up "rapid disease progressors" had the highest levels of angiogenesis (mean rank for MVD-A = 84.7, MVD-B = 82.1, and TVA = 81.1). On multivariate (logistic regression) analysis, factors found to have independent prognostic significance in complete responders (adjusted odd ratio (95% CI, P value)] were: (a) MVD-B grade I [0.134 (0.10-0.16, P < 0.001)], (b) clinical substage A [0.163 (0.12-0.19, P = 0.008)], (c) Bartl's histological stage II & I [0.262 (0.2-0.32, P = 0.021)], (d) MVD-A grade I [0.28 (0.22-0.36, P = 0.03)], (e) beta2 microglobulin levels less than 3,400 ng/dl [0.31 (0.23-0.42, P = 0.04)]. Kaplan-Meier survival analysis for myeloma-related death (n = 16) shows a mean survival time (in months) of 24.75; SE = 3; 95% CI = 21-28. We conclude that MVD (particularly MVD-B) is a very good predictor for the complete response in patients of MM and should be done routinely on bone marrow biopsies.

Angiogenic patterns and their quantitation in high grade astrocytic tumors

The objectives of this study on high grade astrocytic tumors were (i) to establish differences, if any, between grades III & IV tumors among angiogenic parameters, both qualitative and quantitative, and (ii) to correlate angiogenic parameters with proliferation indices, namely T2a and MIB1 labeling indices. Twenty nine consecutive cases of WHO grades III (11) and IV (18) astrocytic tumors diagnosed in the year-2004 were studied, using H&E and CD34, MIB1 and T2a immunostaining by streptavidin biotin technique. Angiogenic patterns were studied and parameters quantitated using Image Pro Plus software (four hotspots) on CD34 immunostained sections to determine intratumoral microvessel density (iMVD), microvascular area (MVA), aspect, mean diameter (MD) and fractal dimension (FD). Main angiogenic patterns of capillary (18) and glomeruloid (9) types were best developed in glioblastomas. Statistically significant differences (P<0.05) were seen between grades III and IV in iMVD, aspect, MD and FD, but not in angiogenic patterns or MVA (P = 0.27). Statistically significant differences (P<0.05) were seen between glioblastomas with glomeruloid vs. capillary types in iMVD and FD, but not in MVA, aspect and mean vessel diameter. T2a values correlated with MIB1 labeling indices (R = 0.965, P<0.001). Intratumoral endothelial MIB1 LI was significantly higher in grade IV as compared to grade III, but did not correlate with angiogenic parameters. No correlation of angiogenic patterns and proliferation indices was noted (R = -0.221, P = 0.26). Limited follow up data showed all recurrent grade IV tumors to be of glomeruloid type. Increased angiogenesis in grade IV, as compared to grade III, astrocytic tumors is characterized by an increased number/density of vessels: an increase in vessels characterized by disproportionate lengthening and likely associated with the infiltrative properties of the tumors; and an increase in pliable, irregularly shaped or structured vessels. In addition, there is a greater frequency of glomeruloid structures indicating inadequate directional migration of the newly formed vessels. The lack of correlation of these angiogenesis parameters with the MIB1 and T2a proliferation indices reflects the complexity of angiogenesis parameters in high grade gliomas. Further studies are needed to determine the usefulness of the angiogenic parameters in the improved diagnosis (grading) and prognosis of astrocytic tumors.

Angiogenesis in nodal B cell lymphomas: a high throughput study

Aim: To assess the biological significance of vascular endothelial growth factor (VEGF) A, VEGF receptor (Flk-1) and cyclooxygenase 2 (COX2) expression with respect to microvessel density (MVD), proliferative activity (Ki-67),...

Tissue factor, angiogenesis and thrombosis in pancreatic cancer

4001 Background: Coagulation proteins are commonly activated in pancreatic cancer and are closely linked to regulation of angiogenesis. We investigated tumor cell expression of tissue factor (TF), the prime initiator of coagulation, and its association with parameters of angiogenesis, venous thromboembolism (VTE) and survival. Methods: Tissue cores from a bi-institutional retrospective series of patients consecutively resected between January 1994 and February 2002 and followed for a median period of 16 months were used to build a pancreatic cancer tissue microarray. TF expression was graded semiquantitatively using immunohistochemistry (IHC)(grade 0:negative, grade 1: 1- 33% positive, grade 2: 34- 66% positive and grade 3: &gt; 66% cells positive) in pancreatic intraductal dysplasia (n=5) and resected pancreatic cancer (n=122). Study endpoints included correlation of TF with VEGF expression by IHC, microvessel density (MVD), clinical VTE and survival in resected patients. Patients with history of VTE, on anticoagulation or with inadequate follow-up were excluded from analysis of VTE outcomes (n=33). Results: TF expression was observed in all specimens with intraductal dysplasia and 108 resected pancreatic cancers (89%) but not in uninvolved pancreas. Sixty-six patients (54%) with resected pancreatic cancer were found to have high TF expression (defined as ≥ grade 2, the median score), and 56 patients (46%) had low or no TF expression. Tumors with high TF expression were more likely to also express VEGF (80% versus 27% with low TF expression, p&lt;0.0001). Tumors with high TF expression had a higher median MVD (8 versus 5/tissue core with low TF expression, p=0.01). Resected patients with high TF expression had a VTE rate of 20% compared to 5.5% in patients with low TF expression (p=0.04). Median survival in tumors with high TF was 17.9 months versus 12.6 months in those with low TF (p=0.16). Conclusions: TF expression appears to occur early in pancreatic cancer pathogenesis. This is the first report describing an association of TF expression with VEGF expression, increased MVD and clinical VTE in resected pancreatic cancer, confirming the linkage of thrombosis and angiogenesis. Targeting TF in pancreatic cancer could affect both neoplastic and thrombotic outcomes. No significant financial relationships to disclose.

A phase II study of ABT-510 for the treatment of metastatic melanoma

8041 Background: ABT-510 is a synthetic peptide analog of thrombospondin-1 demonstrating inhibition of VEGF/bFGF mediated human endothelial cell migration, proliferation, tube formation, cornea-neovascularization as well as inhibition of tumor growth in vivo (B16F10 melanoma). Having been found to be safe in phase I testing, ABT-510 has entered phase II trials. Presented are the results of a phase II study using ABT-510 for the treatment of stage IV melanoma. Methods: A two stage phase II clinical trial was conducted to assess the anti-tumor activity, safety profile and pharmacodynamics of ABT510 in patients with stage IV melanoma. Patients self-administered 100mg of ABT-510 subcutaneously twice/day. Therapy was continued in the absence of excessive toxicity or tumor progression. Primary endpoint was 18 week progression free survival rate. Enrolled were patients at least 18 years of age with measurable (RECIST) metastatic melanoma, ECOG performance status of 0–2 and normal pre-registration blood tests. Exclusion criteria included: cancer therapy less than 4 weeks prior to registration; failure to recover from prior therapy; brain metastases; significant recent bleeding; uncontrolled hypertension; and ongoing anti-coagulation. Pregnant or nursing women were not eligible. Results: Twenty-one patients (67% male) were enrolled with a median age of 55 years. Most patients had M1c disease (80%) and 62% had prior chemotherapy for stage IV melanoma. None of the patients were ineligible/canceled participation. After the first stage of the trial was complete, only 3 of the first 20 patients enrolled were progression-free at 18 weeks. Having not met the minimal clinical efficacy requirement (at least 7 of the first 20 patients progression-free at 18 weeks) the trial was closed to further accrual. Correlative laboratory studies suggested decreases in some of the measured parameters of angiogenesis (VEGFC, circulating endothelial cells) with no impact on immune homeostasis. Conclusions: Single agent ABT-510 therapy administered at 100mg twice/day to patients with previously treated metastatic melanoma did not demonstrate significant clinical efficacy. However, changes in measured parameters of angiogenesis suggest possible clinical efficacy with higher dosing or in combination with cytotoxic therapy. [Table: see text]

Effect of tibolone on tumor angiogenesis parameters in short-term treated women

10519 Background: Large recent studies have shown increased mammographic density, breast pain and a marginally increased risk of breast cancer in women treated with continuous combined estrogen/progestogen (ccEPT), while tibolone has a minimal effect. A placebo-controled clinical trial (STEM), approved by several ethical comities, has been initiated to assess effect and potential mechanism of action of tibolone on breast tumor. We present the results on 12 angiogenesis-related parameters. Methods: 102 women with breast cancer who had to undergo a mastectomy were treated for 2 weeks with tibolone 2.5 mg/day or placebo. RNA was extracted from tumor snap-frozen biopsies (paired before and after treatments) and reverse-transcripted into 2 cDNA batches on which 6 distinct PCRs were performed in duplicate per parameter/sample. A vascular density (VD) index was determined on CD31 specifically stained tumor sections by counting 10 fields per section on 5 distinct sections per biopsy. Results: The validity of the VD evaluation is highlighted by the high correlation coefficient (r = 0.81) between the data within the placebo group at a 2 week interval. In the tumor samples, no change of the VD index was seen. Since it could be argued that VD index could not correctly reflect a modulation of angiogenesis after only 14 days of treatment, we looked at a potential change of the expression of a large array of angiogenesis-related factors (VEGFs, VEGFRs, PAI-1). No modification of the expression in any of these factors was observed. Conclusions: Our data lead to conclude that, in this study, tibolone displays a neutral effect on angiogenesis in breast tumor. [Table: see text]

Antiangiogenic and Antitumor Effects of Bevacizumab in Patients With Inflammatory and Locally Advanced Breast Cancer

Vascular endothelial growth factor (VEGF) is a potent molecule that mediates tumor angiogenesis primarily through VEGF receptor 2 (VEGFR2). Bevacizumab, a recombinant humanized monoclonal antibody to VEGF, was administered to previously untreated patients to evaluate parameters of angiogenesis. Twenty-one patients with inflammatory and locally advanced breast cancer were treated with bevacizumab for cycle 1 (15 mg/kg on day 1) followed by six cycles of bevacizumab with doxorubicin (50 mg/m(2)) and docetaxel (75 mg/m(2)) every 3 weeks. After locoregional therapy, patients received eight cycles of bevacizumab alone, and hormonal therapy when indicated. Tumor biopsies and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) were obtained at baseline, and after cycles 1, 4, and 7. A median decrease of 66.7% in phosphorylated VEGFR2 (Y951) in tumor cells (P = .004) and median increase of 128.9% in tumor apoptosis (P = .0008) were seen after bevacizumab alone. These changes persisted with the addition of chemotherapy. There were no significant changes in microvessel density or VEGF-A expression. On DCE-MRI, parameters reflecting reduced angiogenesis, a median decrease of 34.4% in the inflow transfer rate constant (P = .003), 15.0% in the backflow extravascular- extracellular rate constant (P = .0007) and 14.3% in extravascular-extracellular volume fraction (P = .002) were seen after bevacizumab alone. Bevacizumab has inhibitory effects on VEGF receptor activation and vascular permeability, and induces apoptosis in tumor cells.

Endothelial microparticles affect angiogenesis in vitro: role of oxidative stress

Endothelium-derived microparticles have recently been described as a new marker of endothelial cell dysfunction. Increased levels of circulating microparticles have been documented in inflammatory disorders, diabetes mellitus, and many cardiovascular diseases. Perturbations of angiogenesis play an important role in the pathogenesis of these disorders. We demonstrated previously that isolated endothelial microparticles (EMPs) impair endothelial function in vitro, diminishing acetylcholine-induced vasorelaxation and nitric oxide production by rat aortic rings and simultaneously increasing superoxide production. Herein, using the Matrigel assay of angiogenesis in vitro and a topological analysis of the capillary-like network by human umbilical vein endothelial cells (HUVECs), we investigated the effects of EMPs on formation of the vascular network. All parameters of angiogenesis were affected by treatment for 48 h with isolated EMPs in a concentration of 10(5) but not 10(3) or 10(4) EMPs/ml. The effects included decreases in total capillary length (24%), number of meshes (45%), and branching points (36%) and an increase in mesh area (38%). The positional and topological order indicated that EMPs affect angiogenic parameters uniformly over the capillary network. Treatment with the cell-permeable SOD mimetic Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (Mn-TBAP) partially or completely restored all parameters of angiogenesis affected by EMPs. EMPs reduced cell proliferation rate and increased apoptosis rate in time- and dose-dependent manners, and this phenomenon was also prevented by Mn-TBAP treatment. Our data demonstrate that EMPs have considerable impact on angiogenesis in vitro and may be an important contributor to the pathogenesis of diseases that are accompanied by impaired angiogenesis.

Peroxisome proliferator‐activated receptors in squamous cell carcinoma and its precursors

Peroxisome proliferator-activated receptors (PPARs) mediate several functions that are of interest in carcinogenesis. Although PPARalpha, PPARbeta, and PPARgamma are expressed in multiple human, their expression has not been investigated in non-melanoma skin cancer. We performed a retrospective paired immunohistochemical analysis of normal skin, actinic keratosis (AK), and squamous cell carcinoma (SCC) among 35 individuals. Specimens were considered PPAR immunoreactive when 1% or more of the tumor cells showed clear evidence of immunostaining. Cyclooxygenase-2 (COX-2) expression, the fraction of proliferating endothelial cells, and microvessel density were also evaluated in these samples. PPARalpha immunoreactivity was significantly less likely to occur in SCC and AK than in normal skin of each individual. In contrast to PPARalpha, PPARbeta appeared to be upregulated in (pre)malignant skin lesions. For each individual, the likelihood that normal skin, AK, or SCC was immunoreactive against PPARgamma was comparable. COX-2 immunopositivity was significantly associated with PPARbeta and PPARgamma immunoreactivity. No statistical differences were noted for the angiogenesis parameters and PPARalpha, PPARbeta, or PPARgamma expression, except that the microvessel density was significantly higher among PPARbeta-immunoreactive SCCs compared to that among immunonegative SCCs. Although further research is warranted, these results suggest that PPAR ligands such as fibrates and thiazolidinediones may have chemoprophylactic properties in skin carcinogenesis.

A New Animal Model to Assess Angiogenesis and Endocrine Function of Parathyroid Heterografts In Vivo

It is still a matter of investigation how angiogenesis and restoration of gland perfusion determine graft function after free parathyroid autotransplantation. We provide a new animal model allowing simultaneous and repetitive in vivo assessment of angiogenesis and endocrine function of parathyroid transplants. Fresh human parathyroid tissue from patients with secondary hyperparathyroidism was grafted into dorsal skinfold chamber preparations of athymic nude mice (CD1-nu; n=8). Equivalent pieces of the same human donor specimens were heat-inactivated and served as control grafts (n=7). In all animals receiving parathyroid transplants, intact human parathyroid hormone levels were detectable by species-specific enzyme-linked immunosorbent assay analysis of plasma samples on day 5 after transplantation and increased by 2.5-fold over the observation period (19 days) in contrast with controls. Plasma Ca levels revealed no differences between the groups. On day 5 after transplantation, intravital fluorescence microscopy revealed murine angiogenic microvessels sprouting along nonperfused human donor vessels, and 1 week later functional microvasculature was established in all parathyroid transplants. Histologic analysis revealed well-vascularized endocrine tissue. In contrast, control grafts were necrotic and partly resorbed; they exhibited no angiogenic activity or well-vascularized fat cells indicating fatty degeneration. In addition, species-specific Western blot analysis revealed vascular endothelial growth factor expression of parathyroid transplants rather than functional vessel density as the functional parameter of angiogenesis determining transplant function in vivo. This model may serve to understand mechanisms associated with specific parathyroid transplant angiogenesis and its significance for transplant function to optimize clinical success of autotransplantation in therapy-resistant patients.

Plasma vascular endothelial growth factor and serum soluble angiopoietin receptor sTIE-2 in patients with dural arteriovenous fistulas: a pilot study

Our aim was to correlate concentrations of circulating vascular endothelial growth factor (VEGF) and serum soluble angiopoietin receptor (sTIE-2) before and after endovascular treatment with the grading in human dural arteriovenous fistulas (DAVFs). In ten patients with DAVFs undergoing diagnostic cerebral angiography and endovascular intervention, pre-treatment and post-treatment levels of plasma VEGF and serum TIE-2 were examined in a prospective study design. A total of 32 plasma samples and 19 serum samples was collected from the cubital vein, the arterial sheath before and--if applicable--after intervention. Plasma VEGF and serum Tie-2 levels were measured by standardized ELISA protocols. In eight of ten patients with DAVF increased circulating VEGF levels (elevation of more than mean + 2 SD of published normal values) were found, whereas two patients showed increased sTIE-2 levels. Six of the seven patients treated by endovascular embolization displayed a post-interventional decrease of VEGF values. The serum TIE-2 levels decreased slightly after intervention. Pre-treatment vVEGF levels varied significantly between patients with grades I and II/III fistulas according to the Cognards classification system. Our pilot study suggests that assessment of angiogenesis parameters in patients with DAVFs might correlate with the DAVFs' grade. To support the hypothesis that a change in angiogenic indicators may serve as indicators for a response to therapy, a larger number of patients should be followed for a longer time period.

Celecoxib inhibits angiogenesis by inducing endothelial cell apoptosis in human pancreatic tumor xenografts

Previous studies suggest that antagonists of cyclooxygenases 1 and 2 (COX-1, -2) inhibit angiogenesis in tumor xenografts, but the molecular mechanisms involved remain unclear. Here we characterized the effects of non-selective (indomethacin) and selective (NS398, celecoxib) cyclooxygenase inhibitors on parameters of angiogenesis in human pancreatic adenocarcinoma cells. COX-1 expression was constitutive in 9/9 pancreatic cancer cell lines, whereas COX-2 and cytosolic phospholipase A2 (cPLA2 ) expression were observed in 4/9 cell lines (BxPC3, Capan2, Cfpac1, and L3.6pl). Production of the COX product, prostaglandin E2, correlated with expression of cPLA2 and COX-2 and was blocked by non-steroidal anti-inflammatory drugs (NSAIDs, indomethacin or NS398). In contrast to the findings of others, neither indomethacin nor NS398 affected tumor cell proliferation or secretion of angiogenic factors (VEGF, bFGF, IL-8) at concentrations that produced maximal inhibition of PGE2 production, and higher concentrations increased angiogenic factor production. We also studied the effects of celecoxib in orthotopic L3.6pl xenografts. Immunofluorescence analyses revealed high-level expression of COX-2 in endothelial cells in L3.6pl xenografts that increased following therapy with celecoxib, whereas the tumor cells expressed uniformly low levels of COX- 2. Celecoxib did not decrease tumor-associated VEGF levels in orthotopic human L3.6pl xenografts, but the drug did decrease tumor microvessel density (MVD) and increase apoptosis in tumor-associated endothelial cells in a dose-dependent fashion. Together, our results demonstrate that the anti-angiogeneic effects of NSAIDs in human pancreatic cancer cells are exerted via direct effects on endothelial cells.