Cyclooxygenase-2 inhibition causes antiangiogenic effects on tumor endothelial and vascular progenitor cells.

Tumor angiogenesis is necessary for solid tumor progression and metastasis. Cyclooxygenase (COX)-2 is known to play an important role in cancer growth and invasion, and it activates the signaling pathways controlling cell proliferation, migration, apoptosis, and angiogenesis. COX-2 is reported to be expressed in many cancer cells. Several studies have reported successful treatment of cancer cells with COX-2 inhibitors (COX-2is). However, the effect of COX-2 inhibition on the tumor endothelium remains to be elucidated. Our study shows that COX-2 is expressed in the vasculature of surgically resected human tumors. To investigate the effects of COX-2 inhibition on the tumor endothelium in vitro, we isolated tumor endothelial cells (TECs) from human melanoma and oral carcinoma xenografts in mice, in which we confirmed that tumor growth was suppressed by inhibiting angiogenesis with the COX-2is NS398. COX-2 mRNA was upregulated in TECs compared to normal endothelial cells (NECs). Cell migration and proliferation were suppressed by NS398 in TECs but not in NECs. The effects of NS398 in vivo were consistent with the in vitro results. The number of CD133+ /vascular endothelial growth factor receptor-2+ cells in circulation was significantly suppressed by COX-2 inhibition. In addition, the number of progenitor marker-positive cells decreased in the tumor blood vessels after COX-2i treatment, which suggests that the homing of progenitor cells into the tumor was also blocked. We conclude that NS398 specifically targets both TECs and vascular progenitor cells without affecting NECs.

Isolated tumor endothelial cells maintain specific character during long-term culture.

Tumor angiogenesis is necessary for solid tumor progression and metastasis. Increasing evidence indicates that tumor endothelial cells (TECs) are more relevant to the study of tumor angiogenesis than normal endothelial cells (NECs) because their morphologies and gene expression are different from NECs. However, it is challenging to isolate and culture large numbers of pure ECs from tumor tissue since the percentage of ECs is only about 1-2% and tumor cells and fibroblasts easily overgrow them. In addition, there has been concern that isolated TECs may lose their special phenotype once they are dissociated from tumor cells. In this study, we have successfully purified murine TECs from four different human tumor xenografts and NECs from murine dermal tissue. Isolated ECs expressed endothelial markers, such as CD31, VE-cadherin (CD144), and endoglin (CD105), for more than 3 months after isolation. TECs maintained tumor endothelial-specific markers, such as tumor endothelial marker 8 (TEM8) and aminopeptidase N (APN), as in tumor blood vessels in vivo. In addition, TECs were more proliferative and motile than NECs. TECs showed a higher response to VEGF and higher expression of VEGF receptors-1 and -2 than NECs did. Stem cell antigen-1 was up-regulated in all four TECs, suggesting that they have a kind of stemness. Cultured TECs maintain distinct biological differences from NECs as in vivo. In conclusion, it was suggested that TECs are relevant material for tumor angiogenesis research.

Cytogenetic abnormalities of tumor-associated endothelial cells in human malignant tumors.

Tumor blood vessels are thought to contain genetically normal and stable endothelial cells (ECs), unlike tumor cells, which typically display genetic instability. Yet, chromosomal aberration in human tumor-associated ECs (hTECs) in carcinoma has not yet been investigated. Here we isolated TECs from 20 human renal cell carcinomas and analyzed their cytogenetic abnormalities. The degree of aneuploidy was analyzed by fluorescence in situ hybridization using chromosome 7 and chromosome 8 DNA probes in isolated hTECs. In human renal cell carcinomas, 22-58% (median, 33%) of uncultured hTECs were aneuploid, whereas normal ECs were diploid. The mechanisms governing TEC aneuploidy were then studied using mouse TECs (mTECs) isolated from xenografts of human epithelial tumors. To investigate the contribution of progenitor cells to aneuploidy in mTECs, CD133(+) and CD133(-) mTECs were compared for aneuploidy. CD133(+) mTECs showed aneuploidy more frequently than CD133(-) mTECs. This is the first report showing cytogenetic abnormality of hTECs in carcinoma, contrary to traditional belief. Cytogenetic alterations in tumor vessels of carcinoma therefore can occur and may play a significant role in modifying tumor- stromal interactions.

Inhibitory effects of epigallocatechin-3 gallate, a polyphenol in green tea, on tumor-associated endothelial cells and endothelial progenitor cells.

The polyphenol epigallocatechin-3 gallate (EGCG) in green tea suppresses tumor growth by direct action on tumor cells and by inhibition of angiogenesis, but it is not known whether it specifically inhibits tumor angiogenesis. We examined the anti-angiogenic effect of EGCG on tumor-associated endothelial cells (TEC), endothelial progenitor cells (EPC), and normal endothelial cells (NEC). EGCG suppressed the migration of TEC and EPC but not NEC. EGCG also inhibited the phosphorylation of Akt in TEC but not in NEC. Furthermore, vascular endothelial growth factor-induced mobilization of EPC into circulation was inhibited by EGCG. MMP-9 in the bone marrow plasma plays key roles in EPC mobilization into circulation. We observed that expression of MMP-9 mRNA was downregulated by EGCG in mouse bone marrow stromal cells. In an in vivo model, EGCG suppressed growth of melanoma and reduced microvessel density. Our study showed that EGCG has selective anti-angiogenic effects on TEC and EPC. It is suggested that EGCG could be a promising angiogenesis inhibitor for cancer therapy.

Adrenomedullin antagonist suppresses tumor formation in renal cell carcinoma through inhibitory effects on tumor endothelial cells and endothelial progenitor mobilization.

Adrenomedullin (AM) is a multifunctional 52-amino acid peptide. AM has several effects and acts as a growth factor in several types of cancer cells. Our previous study revealed that an AM antagonist (AMA) suppressed the growth of pancreatic tumors in mice, although its mechanism was not elucidated. In this study, we constructed an AMA expression vector and used it to treat renal cell carcinoma (RCC) in mice. This AMA expression vector significantly reduced tumor growth in mice. In addition, microvessel density was decreased in AMA-treated tumors. To analyze the effect of AMA on tumor angiogenesis in this model, tumor endothelial cells (TECs) were isolated from RCC xenografts. TEC proliferation was stimulated by AM and it was inhibited by AMA significantly. AM induced migration of TECs and it was also blocked by AMA. However, normal ECs (NECs) were not affected by either AM or AMA. These results demonstrate that AMA has inhibitory effects on TECs specifically, not on NEC, thereby inhibiting tumor angiogenesis. Furthermore, we showed that vascular endothelial growth factor-induced mobilization of endothelial progenitor cell (EPC) into circulation was inhibited by AMA. These results suggest that AMA can be considered a good anti-angiogenic reagent that selectively targets TECs and EPC in renal cancer.