Quantitation of angiogenesis and its correlation with vascular endothelial growth factor expression in astrocytic tumors

OBJECTIVE: To quantitate tumor angiogenesis by establishing intratumoral microvessel density (IMD), to study vascular endothelial growth factor (VEGF) expression in different grades of astrocytomas and to correlate VEGF expression with tumor angiogenesis. STUDY DESIGN: Forty cases of astrocytic neoplasms (10 of each grade) were assessed for tumor angiogenesis and VEGF expression. The panendothelial marker CD31 was used to highlight microvessels. Tumor angiogenesis was quantitated as IMD count per square millimeter in areas of high vascularity, or "hot spots," using an image analyzer. VEGF expression was studied in sections of the tumors. IMD counts per square millimeter and VEGF expression were correlated with histologic grade. The angiogenic potential of tumors as reflected by IMD counts per square millimeter was correlated with the intensity of VEGF expression. RESULTS: Vascular proliferation in high grade gliomas was significantly higher as compared to that in low grade gliomas. IMD count per square millimeter revealed a positive correlation with histologic grade in high grade gliomas. Pilocytic astrocytoma and low grade astrocytoma as a group had comparable IMD counts per square millimeter. VEGF expression paralleled IMD counts in rare high grade gliomas only. CONCLUSION: Malignant progression in astrocytoma is heralded and accompanied by increased angiogenesis. VEGF is an important angiogenic factor in high grade gliomas since its expression parallels the increased IMD counts in these tumors. In contrast, in low grade gliomas, angiogenic factors other than VEGF may contribute to vascular proliferation. The results emphasize the role of antiangiogenic therapy as an optimal tool in therapeutic strategies as they become available.     

Loss of heterozygosity at chromosomal regions 3p and 13q in non-small-cell carcinoma of the lung represents low-frequency events

It was recently reported that loss of heterozygosity occurred at the chromosomal region 3p in small-cell as well as in non-small-cell carcinoma of the lung. A recent report also indicated genetic changes involving sequences on chromosomes 13q and 17p in small-cell and in non-small-cell carcinomas. In the present study normal and tumor DNAs representing mostly adeno-and squamous cell carcinomas of the lung were examined for loss of heterozygosity on chromosomes 3p, 13q, 11p, and 1p. With the exception of two non-small-cell carcinomas which demonstrated loss of alleles on chromosome 3p and one small-cell carcinoma which demonstrated loss of heterozygosity at chromosome 3p as well as at 13q, evidence for loss of alleles on chromosomes 3p, 13q, 11p, and 1p could not be obtained in greater than 75% of the non-small-cell carcinoma DNAs tested. Given this result it appears unlikely that a recessive gene is located on either chromosome 3p or 13q in the majority of non-small-cell carcinomas of the lung.     

Playing only one instrument may be not enough: limitations and future of the antiangiogenic treatment of cancer

Angiogenesis plays an essential role in tumor growth, invasion and metastasis. After initial pessimism about the usefulness of the antiangiogenic therapeutic approach for cancer, interest has increased in the development of antiangiogenic compounds after the first clinical approval of an antiangiogenic therapy. The anti-vascular endothelial growth factor (VEGF) antibody bevacizumab has recently been approved for use in combination with chemotherapy for the treatment of metastatic colorectal and non-small cell lung cancer patients. However, no survival benefit has been demonstrated in anti-VEGF monotherapy trials, probably due to the high complexity of tumor angiogenesis regulation. Experimental and clinical data, including the approval of the multitargeted drugs sunitinib and sorafenib, indicate that exciting results, including tumor regression, can be expected from the combined targeting of different pathways in the tumor angiogenesis scenario. Several obstacles, including the high cost of new molecular targeted drugs make this therapeutic approach difficult.     

Relationship between vascular endothelial growth factor and angiogenesis in spontaneous and indomethacin-delayed healing of acetic acid-induced gastric ulcers in rats.

Angiogenesis is an important event for gastric ulcer healing. Vascular endothelial growth factor (VEGF) is known to be a potent stimulator of angiogenesis. This study consequently examined VEGF production, VEGF mRNA expression and angiogenesis during the spontaneous and indomethacin-delayed healing of acetic acid-induced ulcers in rats. The production of VEGF, taking place in the normal mucosa, was significantly elevated by ulceration. The mRNA expression of three isoforms of VEGF (VEGF188, VEGF164 and VEGF120) was also detected. Following the increase in VEGF production, angiogenesis was significantly promoted in the ulcer base. VEGF-immunoreactivity was observed in granulocytes, fibroblasts and regenerated epithelial cells. Indomethacin markedly inhibited prostaglandin E2 synthesis in the ulcer base, resulting in the prevention of ulcer healing. Angiogenesis was also significantly inhibited by indomethacin, but neither VEGF production nor VEGF mRNA expression was reduced. Such results suggest that VEGF might play a role in angiogenesis in the spontaneous healing of gastric ulcers in rats. However, the inhibition of angiogenesis in indomethacin-delayed ulcer healing is not explainable on VEGF expression.     

Expression of vascular endothelial growth factor and basic fibroblast growth factor receptors in lung cancer

OBJECTIVE: To determine the expression of two angiogenic factors, vascular endothelial growth factor (VEGF) and fibroblast growth factor receptors (FGFR), in non-small cell lung carcinoma (NSCLC) in relation to tumor stage (TN0, TN1, TN2) and in association with the expression of p53 protein, a potential suppressor of tumor angiogenesis. STUDY DESIGN: The immunohistochemical (IHC) expression of VEGF and FGFR was examined in paraffin sections of 56 NSCLC in relation to the presence of lymph node metastases and p53 expression. Nodal status of NSCLC determined: 27 tumors, N0; 16, N1; and 13, N2 stage. Semiquantitative analysis with a score corresponding to IHC staining intensity and percentage of positive cells was used. Statistical analysis was performed with the chi 2 test. RESULTS: A significant association was noted between VEGF and FGFR expression in NSCLC. No relation was found between VEGF, FGFR expression and lymph node metastasis or p53 expression. CONCLUSION: We assume that VEGF and FGFR act in a synergistic manner in NSCLC and that their expression is not related to lymph node metastases. Angiogenesis is a very complex phenomenon and heterogeneous within tumors. Also, it is affected by microenviromental factors.     

Hyaluronic acid as an anti-angiogenic shield in the preovulatory rat follicle

Angiogenesis in the preovulatory follicle is confined to the theca cell layers, and penetration of capillaries through the basement membrane into the granulosa cell layers does not occur until after ovulation. However, elevated expression of the angiogenic growth factor (VEGF) has been reported in the cumulus cells surrounding the oocyte, which are expelled from the follicle during ovulation. This spatial and temporal discrepancy between VEGF expression and angiogenesis was studied here in the rat ovarian follicle, and we showed that cumulus cells secrete to the follicular fluid, in addition to VEGF, material with antiangiogenic activity that blocks endothelial cell proliferation, migration, and capillary formation in vitro. Hyaluronic acid produced by the cumulus cells can account for this antiangiogenic activity. Degradation of hyaluronic acid by hyaluronidase restored proliferation and migration of endothelial cells directed toward the cumulus. Inhibition of hyaluronic acid synthesis with 6-diazo-5-oxo-1-norleucine restored endothelial proliferation and migration in vitro, and it also resulted in early penetration of capillaries across the follicular basement membrane in vivo. These results support the role of hyaluronic acid produced by the cumulus cells as a high-molecular-weight, antiangiogenic shield that prevents premature vascularization of the preovulatory follicle by blocking endothelial cell migration and proliferation.     

Is vasohibin-1 for more than angiogenesis inhibition?

Angiogenesis, a formation of neo-vessels from pre-existing ones, is regulated by the local balance between its stimulators and inhibitors. Vasohibin-1 (VASH1) was originally identified as an endothelium-derived vascular endothelial growth factor (VEGF)-inducible angiogenesis inhibitor that acts in a negative feedback manner. The expression of VASH1 has been shown in endothelial cells (ECs) in both physiological and pathological conditions associated with angiogenesis. However, recent reports indicate that VASH1 is expressed not only in ECs but also in other cell types including haematopoietic cells. The function of VASH1 may not be restricted to angiogenesis inhibition.     

Role and mechanism of arsenic in regulating angiogenesis

Arsenic is a wide spread carcinogen associated with several kinds of cancers including skin, lung, bladder, and liver cancers. Lung is one of the major targets of arsenic exposure. Angiogenesis is the pivotal process during carcinogenesis and chronic pulmonary diseases, but the role and mechanism of arsenic in regulating angiogenesis remain to be elucidated. In this study we show that short time exposure of arsenic induces angiogenesis in both human immortalized lung epithelial cells BEAS-2B and adenocarcinoma cells A549. To study the molecular mechanism of arsenic-inducing angiogenesis, we find that arsenic induces reactive oxygen species (ROS) generation, which activates AKT and ERK1/2 signaling pathways and increases the expression of hypoxia-inducible factor 1 (HIF-1) and vascular endothelial growth factor (VEGF). Inhibition of ROS production suppresses angiogenesis by decreasing AKT and ERK activation and HIF-1 expression. Inhibition of ROS, AKT and ERK1/2 signaling pathways is sufficient to attenuate arsenic-inducing angiogenesis. HIF-1 and VEGF are downstream effectors of AKT and ERK1/2 that are required for arsenic-inducing angiogenesis. These results shed light on the mechanism of arsenic in regulating angiogenesis, and are helpful to develop mechanism-based intervention to prevent arsenic-induced carcinogenesis and angiogenesis in the future.     

Immunocytologic diagnosis of small-cell lung cancer in imprint smears.

Imprints of histologic or autopsy specimens from 12 small-cell lung cancers (SCLCs), 82 non-SCLCs (50 adenocarcinomas, 25 squamous-cell carcinomas, 1 adenosquamous carcinoma and 6 large-cell carcinomas), 2 carcinoid tumors, 1 malignant lymphoma and 8 metastatic carcinomas were examined immunocytologically for the presence of cluster 1 SCLC antigen (neural-cell adhesion molecule: N-CAM), chromogranin A, Leu-7, neuron-specific enolase (NSE) and gastrin-releasing peptide (GRP). The monoclonal antibodies NCC-LU-243 and NCC-LU-246, which are reactive with cluster 1 SCLC antigen/N-CAM, diffusely stained the cell membranes of all SCLCs and carcinoid tumors (100%) and diffusely and focally stained those of two of the large-cell carcinomas, two of the adenocarcinomas, two of the squamous-cell carcinomas and the one adenosquamous carcinoma. Malignant lymphoma and metastatic carcinoma were negative for this antigen. A few cases of large-cell carcinoma, adenocarcinoma, squamous-cell carcinoma and adenosquamous carcinoma were also stained with these antibodies, which may indicate a neuroendocrine differentiation. However, these tumors were different from SCLCs in that their positive tumor cell population was definitely smaller than that in SCLC, in which almost all tumor cells were positive. This confirmed the usefulness of antibodies against cluster 1 SCLC antigen for the immunocytologic diagnosis of SCLC and carcinoid tumor in imprint smears. Chromogranin A, GRP, NSE and Leu-7 were not useful in immunocytologically differentiating the imprints from these cases since only a few tumor cells were reactive with these antibodies. The antibodies against cluster 1 SCLC antigen/N-CAM can also be applied to cytologic preparations of sputum, pleural fluid and fine needle aspirates stained routinely by the Papanicolaou method since the antigen is preserved in such alcohol-fixed smears.     

Poster Sessions BP02: Oligodendrocytes and Schwann Cells

Neurofibromatosis Type 1 tumors are highly vascularized and contain Schwann cells with hyperactivated Ras. In vitro , the NF1-derived neurofibromin deficient Schwann cells have an angiogenic profile, which favors angiogenesis and sustains the growth of the NF1-derived tumors. This study examined the relationship of the activation state of Ras as it related to the expression of angiogenic and antiangiogenic factors in both cultured NF1-derived Schwann cells and normal human Schwann cells. Western blot analysis of normal human Schwann cells revealed low expression of angiogenic vascular endothelial growth factor (VEGF) as well as low expression of the antiangiogenic pigment epithelium derived factor (PEDF). Relative to normal human Schwann cells, NF1-derived Schwann cells have increased RAS activity and a three-fold increase in VEGF expression. Surprisingly, PEDF was also expressed in the NF1-derived Schwann cells at approximately the same level as VEGF expression. Using a retroviral construct, we introduced the GAP-related domain of neurofibromin into the NF1-derived Schwann cells to reduce the level of activated Ras. Relative to the untreated NF1-derived Schwann cells the Schwann cells expressing the GAP-related domain expressed about one-half the VEGF but twice the PEDF. We conclude that decreasing the Ras activity in NF1-drived Schwann cells will not only decrease proliferation, but also slow tumor angiogenesis due to the decreased expression of angiogenic and increased expression of antiangiogenic factors.     

Thrombospondin and vascular endothelial growth factor are cyclically expressed in an inverse pattern during bovine ovarian follicle development

Angiogenesis does not normally occur in most adult tissues. However, in the ovary, there are cyclical vascular changes including angiogenesis that involve the interaction of numerous cytokines and growth factors. Angiogenic processes are regulated by a balance between pro- and antiangiogenic factors. The purpose of this study was to determine the expression of the antiangiogenic thrombospondin family and proangiogenic vascular endothelial growth factor (VEGF) in various sizes of healthy bovine follicles. Ovaries were collected from slaughterhouse animals and healthy follicles were sorted based on size (< 0.5 cm, small; 0.5-1.0 cm, medium; >1.0 cm, large). Thrombospondin (TSP) protein levels were significantly higher in small follicles. Immunohistochemistry confirmed the granulosa layer as the primary area within the follicle involved in TSP generation and that small follicles had the highest proportion of immunopositive cells. TSP-1 and -2 mRNA levels were significantly higher in small follicles than either medium or large follicles. TSP colocalized with CD36 on granulosa cells (GC) in the follicle and in cultured cells. In contrast with TSP, VEGF expression increased during growth and development of the follicle. FSH stimulated GC expression of TSP, while LH had no effect. In summary, TSP-1 and -2 were coordinately expressed in the extravascular compartment of the ovary during early follicle development. VEGF was inversely expressed, with expression increasing as follicles developed. Regulated expression and localization of these proteins suggests that they may be involved in regulating growth and development of the follicle in a novel fashion.     

Quantitative RT-PCR assay for VEGF mRNA in human tumors of the kidney

Angiogenesis is the formation of new capillaries from pre-existing vessels, and recent evidence has demonstrated that tumor growth is controlled mainly by angiogenesis. Vascular endothelial growth factor (VEGF) is an endothelium-specific growth factor which is strongly angiogenic in vitro and in vivo. We have developed a quantitative RT-PCR assay for the measurement of VEGF mRNA expression using a real-time procedure based on the use of fluorogenic probes and the ABI PRISM 7700 Sequence Detector System. The assay performance of this method in terms of practicability and reliability is reported with results that seem promising for its widespread use in the clinical laboratory. The method has been applied to the measurement of mRNA of VEGF in human renal cell carcinomas (RCC). Preliminary results show a significantly higher VEGF mRNA expression (ratio values between 181 and 2222) in tumor specimens compared to non-adjacent, non-tumoral tissue of the same subjects.     

Poster Sessions BP02: Oligodendrocytes and Schwann Cells. RAS activation regulates angiogenic and anti‐angiogenic factor expression in NF1‐derived Schwann cells

Neurofibromatosis Type 1 tumors are highly vascularized and contain Schwann cells with hyperactivated Ras. In vitro, the NF1‐derived neurofibromin deficient Schwann cells have an angiogenic profile, which favors angiogenesis and sustains the growth of the NF1‐derived tumors. This study examined the relationship of the activation state of Ras as it related to the expression of angiogenic and antiangiogenic factors in both cultured NF1‐derived Schwann cells and normal human Schwann cells. Western blot analysis of normal human Schwann cells revealed low expression of angiogenic vascular endothelial growth factor (VEGF) as well as low expression of the antiangiogenic pigment epithelium derived factor (PEDF). Relative to normal human Schwann cells, NF1‐derived Schwann cells have increased RAS activity and a three‐fold increase in VEGF expression. Surprisingly, PEDF was also expressed in the NF1‐derived Schwann cells at approximately the same level as VEGF expression. Using a retroviral construct, we introduced the GAP‐related domain of neurofibromin into the NF1‐derived Schwann cells to reduce the level of activated Ras. Relative to the untreated NF1‐derived Schwann cells the Schwann cells expressing the GAP‐related domain expressed about one‐half the VEGF but twice the PEDF. We conclude that decreasing the Ras activity in NF1‐drived Schwann cells will not only decrease proliferation, but also slow tumor angiogenesis due to the decreased expression of angiogenic and increased expression of antiangiogenic factors.     

Atorvastatin sensitizes human non-small cell lung carcinomas to carboplatin via suppression of AKT activation and upregulation of TIMP-1

Platinum-based chemotherapy is the standard treatment for advanced non-small-cell lung carcinomas (NSCLCs). However, the antitumoral effect of carboplatin displays unsatisfactory in NSCLCs treatment due to the AKT pathway-mediated carboplatin insensitive in NSCLCs treatment. Previous studies have shown that statins have antitumor activity, but it is unknown whether atorvastatin can reverse carboplatin resistance in lung cancer. Treatment with atorvastatin and carboplatin reduced the growth of xenograft A549 tumors in nude mice and enhanced the survival rate compared with carboplatin alone. Atorvastatin in combination with carboplatin had stronger effects on growth inhibition and apoptosis of NSCLC than either agent used individually. Carboplatin conferred anti-invasive effect in NSCLC cells mainly through inhibition of AKT activity and resultant upregulation of TIMP-1. However, the inhibitory effect on AKT activity by carboplatin was short-term. Additional atorvastatin administration resulted in synergistic inhibition of NSCLC cell invasion and stimulation of TIMP-1 expression with carboplatin through stronger and persistent inhibition of AKT activity both in vivo and in vitro. The synergy of atorvastatin and carboplatin was confirmed using another human lung carcinoma cell line (H1299). Altogether, our data demonstrate that atorvastatin may overcome carboplatin resistance in lung cancer by suppressing AKT activity and upregulating TIMP-1. A combination of atorvastatin and carboplatin may be an effective strategy in clinical therapy against NSCLCs.