| Objectives:To observe the effects of recombinant human endostatin (rh-endostatin) on kinetics and viability of circulating endothelial cells(CECs). tumor vasculature and tumor angiogenesis-related factors, to explore the surrogate markers corresponding with the "normalization window", and to investigate the optimizing schedule of combination therapies on tumor growth.Method:Nude mice were randomized into3groups for mono-therapy, control group (NS200μl·d-1.14days), rh-endostatin group (rh-endostatin20mg·kg-1·d-1,14days) and cisplatin group (cisplatin4mg·kg-1·d-1. days1to5). Nude mice were randomized into4groups for combined therapy, control group (NS200μl·d-1,14days), simultaneous combined group (rh-endostatin20mg·kg-1·d-1, days1to14; cisplatin4mg·kg-1·d-1days1to5). rh-endostatin plus cisplatin group(rh-endostatin20mg·kg-1·d-1, days1to14; cisplatin4mg·kg-1·d-1. days5to9) and cisplatin plus rh-endostatin group(cisplatin4mg·kg-1·d-1. days1to5:rh-endostatin20mg·kg-1·d-1. days6-19). The largest diameter and the vertical diameter of tumors were measured at different time points. CECs were enumerated by flow cytometry (FC). kinetic changes in vascular morphology were detected under a confocal microscope with immunofluorescent staining in carcinomas in mice. and the vascular permeability was quantified by the amount of diffused dextran by Image Pro Plus6.0software. And we monitored EMMPRIN, VEGF, MMP2, MMP9, TIMP-1, TIMP-2, HIF-1α, microvascular density (MVD), and collagen coverage of tumor vessels at different time points by immunohistochemical staining.Results:1. The results of animal experiment:Both rh-endostatin (182.408mm3±46.379mm3) and cisplatin (234.774mm3±59.588mm3) mono-therapies inhibited tumor growth. All combined therapies inhibited tumor growth significantly. There was no significant body weight difference among groups (P>0.05).2. The results of evaluation of CECs by FC:After rh-endostatin administration, aCEC was increased on day7(P=0.003) and decreased on day10(P=0.026), and apoptotic CECs was increased on day10(P=0.033). Rh-endostatin plus cisplatin group and cisplatin plus rh-endostatin group led to a significant decrease in aCECs. All combined therapy groups led to a significant increase in apoptotic CECs. 3. Tumor vascular permeability decreased by rh-endostatin:The tumor vasculature in rh-endostatin group had fewer giant branches, blood vessel tortuosity and dilation decreased, which significantly alleviated vascular permeability on days7and10.4. The comparison of MVD and collagen coverage of tumor vasculature:MVD was decreased and the ratio of collagen coverage of vessels was increased respectively on days7and10after rh-endostatin administration. MVD was decreased and the ratio of collagen coverage of vessels was increased in all combined therapy groups.5. The results of immunohistochemistry staining:After rh-endostatin administration, HIF-1α was decreased on day7. EMMPRIN, VEGF, MMP-2and MMP-9were all decreased on day4. TIMP-1and TIMP-2were increased on days4and7. Tumor oxygenation was improved in rh-endostatin plus cisplatin group and cisplatin plus rh-endostatin group. EMMPRIN, VEGF. MMP-2and MMP-9were all down-regulated and TIMP-2was up-regulated in all combined therapy groups.6. The correlation analysis:A positive correlation was found between HIF-la. EMMPRIN. VEGF. MMPs and MVD. and between HIF-1α and tumor volume. A negative correlation was found between VEGF. MMP-9, HIF-1α and TIMP-1and TIMP-2. A negative correlation was found between collagen coverage of vessels and MVD. VEGF, HIF-1α; MMP-2and MMP-9. a positive correlation was found between collagen coverage of vessels and TIMP-1and TIMP-2.Conclusion:Rh-endostatin could transiently normalize tumor vasculature and microenvironment on days4to10after administration probably via restoration of the balance between pro-and anti-angiogenesis factors. During the time of vascular normalization, the combined treatment was found to have maximal effect on tumor growth delay. The change of CECs should be a dynamic process with increased aCECs in early-stage suggesting the decrease of vascular bed and the start of vascular normalization, and sequent decline in aCECs and increased apoptotic CECs reflecting the decreased capacity of angiogenesis and the close of the vascular normalization window after rh-endostatin therapy. The enumeration of CECs could be a useful biomarker for defining "vascular normalization window" and providing important basis for making optimizing combined therapy schedule in human tumor. |
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