| PART ? Differential effects of endostar treatment on differentiated and undifferentiated blood vessels in Lewis lung CancerBackgroundAngiogenesis is crucial to tumor growth and is regarded as an important target for cancer therapy. A number of anti-angiogenic drugs have been developed, including monoclonal antibodies (mAbs) and synthetic tyrosine kinase inhibitors. Anti-angiogenic agents have been approved to be used in various types of cancer and many patients have benefited from these angiogenesis inhibitors in preclinical or clinical study.Microvessel density is the current reference standard for characterization of tumor angiogenesis. Different blood vessel markers are used to reveal different aspects and characteristics of tumor vasculature. CD31 microvessel immunostaining has advantages as a sensitive method for all microvessels include undifferentiated or differentiated ones. While CD34 shows a high sensitivity for the staining of normal vascular endothelium, the specificity is restricted to differentiated, well-formed vessels.Endostatin, as an endogenous anti-angiogenic protein, played a crucial role in suppression of angiogenesis and tumor growth in many tumors including Lewis lung cancer. Much evidence suggests that anti-angiogenesis transiently normalized tumor vasculature in tumors. Normalization of the tumor vasculature and microenvironment by a rh-endostatin (endostar) has also been reported in Lewis lung carcinoma. However the mechanism and significance of "vessel normalization" is not clear.The factors involved in different ways of angiogenesis does not completely consistent, and the differentiation of new blood vessels formed in different ways also has different. Any anti-angiogenesis drug we used mostly role on a species molecular or a special form in the formation of new blood vessels. So the drug may have different effect on the different ways of angiogenesis and also different effect on different differential new blood vessels.ObjectiveAngiogesis is stated in both physiological and pathogenesis processes. There were both differentiated (CD34+) and undifferentiated (CD31+/CD34-) blood vessels in the angiogesis process. In this study, we evaluated the different effects of endostar on different differential tumor vasculature in Lewis lung cancer.MethodsSubcutaneous Lewis lung carcinomas were established in mice, which were treated by different doses of endostar or physiological saline daily for 14 days. The volume of the tumor was recorded daily and the status of the mice was assessed. The intravital microscopy was performed at the 7th day after first treatment. Tumor specimens were stained with hematoxylin and eosin(H&E) and expression of CD31 and CD34, the different endothelial cell markers, was determined by immunohistochemical straining. The relationship between different doses of the drug and the level of microvessel density (MVD) was analyzed.Results1 The tumor volumes of different doses of endostar groups were smaller than those of the control group (P< 0.05). Tumor growth was inhibited by the higher doses of endostar and 50mg/kg endostar had the most distinct tumor inhibition.2 Endostar normalizes the architecture of the vascular network more like normal vessels in skeletal muscle was observed through two-photon confocal confocal microscopy.3 Marked vasculature CD31 immunofluorescence was more significant than CD34 immunofluorescence labeling of blood vessels. Compared to the control group, the MVD value of CD31 in endostar treatment groups decreased, especially in the 50mg/kg group (P< 0.05). However, the MVD value of CD34 in endostar treatment groups did not decrease, even in the 50mg/kg group (P>0.05).ConclusionUndifferentiated MVD was significantly reduced by different doses of endostar treatment, and with increasing doses the decline was more evident. This may be the reason for less toxity for host and normalization of tumor blood vessels by endostar.PART II Assessment of hemodynamic changes in Lewis lung cancer treated by endostar:using quantitatively contrast--enhanced ultrasoundBackgroundAnti-angiogenic agents have been approved to be used in various types of cancer and many patients have benefited from these angiogenesis inhibitors in preclinical or clinical study. Endostatin played a crucial role in suppression of endothelial proliferation, angiogenesis, and tumor growth in many tumors. Normalization of the tumor vasculature and microenvironment by Endostar has been reported in Lewis lung carcinoma.There is tremendous interest in the non-destructive imaging methods for tumor angiogenesis, such as contrast-enhanced dynamic computed tomography (CT) or magnetic resonance imaging (MRI), positron emission tomography, and so on. Ultrasound(US) is also an emerging platform technology for this goal. Contrast-enhanced ultrasonography is more sensitive in slow blood flow than other imaging methods, therefore it can offer more detailed information of tumor vascularity.Microvessel density is the current reference standard for characterization of tumor angiogenesis. Different blood vessel markers are used to reveal different aspects and characteristics of tumor vasculature. Contrast-enhanced ultra sonography(CEUS) is not only applicable for assessment of vascular quantity but also be suitable as an indicator of blood flow changes in vasculature normalization during the period of endostar treatment. There is a complex relationship between the ultrasound imaging hemodynamic parameters and microvessel.Normalization of tumor vasculature by endostar was reported. Contrast-enhanced ultrasound(CEUS) is emerging as a worthful methods for vascular imaging indications for hemodynamic change in tumor blood vessel by endostar treatment.ObjectiveWe assessed the dynamic changes of hemodynamic parameter in Lewis lung carcinoma treated with endostar by CEUS.MethodsSubcutaneous Lewis lung carcinomas were established in mice, which were treated by 5mg/kg endostar or physiological saline daily for 14 days. Ultrasound imaging using a 15L8-MHz linear array probe at 2,5,8,11 and 14 days after the first treatment and we used sonovue as the ultrasound contrast agent for examinations to analyse dynamic perfusion parameters(vascular patterns) in the contrast process. After the last time of CEUS, the minces were euthanized and tumour tissue were taken out for fixation. Tumor specimens were stained with hematoxylin and eosin(H&E) and expression of CD31 was determined by immunohistochemical straining. The relationship between different hemodynamic parameters PI, mTT, TTP at the last time and the level of MVD was analyzed.ResultsThe tumor volumes of experimental group were smaller than those of the control group (P< 0.05). Some quantitative parameters differed between the groups at different time. Peak Index(PI) was decreased in treatment group at day 11(25.89±10.27dB vs 35.17±12.33 dB, P<0.05), however Time to Peak(TP) was extended during day 8 to day 11(10.54±5.36 vs 6.54±4.23 at day 8, P<0.05), and Mean Transit Time(mTT) of perfusion was shorter during day 5 to day 8(59.35±11.22 vs 82.25±11.36 at day 5, P<0.05).The mean MVD values of CD31+of control and treatment groups were 72.93 ± 12.20 and 54.07± 10.76, respectively. There was a positive correlation between PI and CD31 MVD values in Lewis Lung carcinoma, which was statistically significant (r= 0.692; P<0.05). Other parameters:Time to Peak (TTP), mean Transit Time (mTT) had no obvious correlation with the microvessel density (P>0.05).ConclusionsIn conclusion, We confirmed CEUS imaging is suitable to assess the early efficacy of endostar treatment on Lewis lung cancer. And we suggested endostar caused microvascular normalization resulting in changes of blood flow velocity before the drug significantly reduced the number of microvessels... |
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