| Part 1 The expression of adrenomedullin in the renal tumor tissue and its relationship with the microvessel densityObjective: To explore the role of ADM in the development of the renal cell carcinoma and the angiogenesis in tumor of kidney and provide a molecular biology target, which could study the invasion and metastasis, prognosis and staging of molecular biology of renal cell carcinoma, the expression of the adrenomedullin (ADM) in the renal cell carcinoma and normal kidney tissue was detected by immunohistochemistry and flow cytometry. The microvessel density (MVD) in renal cell carcinoma and normal kidney tissue was further detected. This study provided the theory basis of the comprehensive treatment, especially the gene targeting therapy of the renal cell carcinoma.Method: We studied 33 operation cases with renal carcinoma. Draw materials from the specimen immediately after ex vivo, including the tumor tissue and the normal renal parenchyma tissue whose distance to the tumor edge over 2cm. The expression of ADM, ADMR and MVD were detected by immunohistochemistry and flow cytometry. Correlation analysis was performed according to the expression of ADM, ADMR and MVD.Results: Immunohistochemistry analysis showed that the positive cells of ADM were 100% in renal cell carcinoma cases, while weakly positive expression of ADM was 9.1% in the normal kidney group. The expression of ADM tested by flow cytometry in the renal cell carcinoma tissues (1267.84±127.52) was evidently higher than that in the normal kidney tissues (970.36±171.46) (P=0.000<0.05). Meanwhile, the expression level of the ADMR by flow cytometry in the renal cell carcinoma tissues (1161.91±202.71) was evidently higher than that in the normal kidney tissues (826.05±171.99) (P=0.000<0.05). Theses data showed that the expression of ADM and ADMR with MVD in the renal cell carcinoma was significantly positively correlated. Conclusion: The expression of ADM and ADMR in the renal cell carcinoma was obviously higher than that in the normal kidney tissues, which positively correlated with the MVD of renal cell carcinoma. These data indicate that ADM may promote the formation of neovessels of the kidney tumor, suggesting it may be a new therapy target of renal cell carcinoma.Part II The effect of hypoxia on the expression of ADM and the effect of ADM on the expression of VEGF in renal tumor cellsObjective: To further explore the role of ADM in renal tumor angiogenesis,the expression of ADM induced by hypoxia and the expression of vascular endothelial growth factor (VEGF) induced by ADM in renal tumor cells were detected by Western blot and RT-PCR.Method: Human renal tumor cells 786-o were cultured respectively in low-oxygen-partial-pressure environment (5% CO2, 94.55% N2, 0.45% O2, 37℃) and normal oxygen-partial-pressure environment (20% O2, 5% CO2, 37℃). The expression of ADM in renal tumor cells was detected by Western boltting and RT-PCR. The expression of VEGF treated by ADM or/and ADM receptor antagonist in normal oxygen-partial-pressure environment in 786-o cells was detected by Western boltting and RT-PCR.Results:1 Hypoxia increased the expression of ADM in renal tumor cellsRenal tumor cells 786-o were cultured for 3 hours in hypoxia environment. The ADM expression in renal tumor cells was significantly increased compared with the control group (t=-8.603, P=0.000<0.05) in time-dependent manner. The ADM mRNA expression in renal tumor cells was significantly higher than the control group (t=-13.277, P=0.000<0.05) in time-dependent manner. 2 The effect of ADM on VEGF expression in renal tumor cells.The expression of VEGF in renal tumor cells group treated with ADM was significantly increased compared with the control group? in a time-dependent manner (24h t=-3.069 P=0.007<0.05; 48h t=-18.692 P=0.000<0.05; 72h t=-35.184 P=0.000<0.05). There is no significant difference in the expression of VEGF between the ADM+ADM receptor inhibitor group and the control group (24h t=0.922 P=0.369>0.05; 48h t=0.043 P=0.966>0.05; 72h t=-0.372 P=0.714>0.05).RT-PCR showed that VEGF mRNA in renal tumor cells treated by ADM was increased compared with the control group in a time-dependent manner (24h t=-2.910 P=0.009<0.05; 48h t=-19.006 P=0.000<0.05; 72h t=-30.973 P=0.000<0.05). There is no significant difference in the level of VEGF mRNA between the ADM+ADM receptor inhibitor group and the control group (t=-0.597 P=0.558>0.05; 48h t=0.605 P=0.553>0.05; 72h t=-0.620 P=0.543>0.05).Conclusion: Hypoxia can induce the expression of ADM in human renal tumor cells in a time-dependent manner. ADM increase the expression of VEGF in renal tumor cells. ADM receptor inhibitor can effectively interrupt the expression of VEGF induced by ADM in renal cell carcinoma. In conclusion, ADM may regulate the process of angiogenesis through increasing the expression of VEGF in renal tumor.Part III The effect of ADM on the signal transduction pathway of the renal tumor angiogenesisObjective: PI3K/Akt signaling pathway can regulate cell growth, angiogenesis and tolerance with hypoxia or nutritional deficiency. To evaluate the activation of PI3K/Akt pathway induced by ADM in renal tumor cells and the effect of ADM on the expression of eNOS and its effect on angiogenesis and progression in renal tumor, Western blot was performed to detect the expression of eNOS and signaling molecules.Method: Human renal tumor 786-o cells were divided into five groups, the control group, the ADM group, the VEGF group, the ADM+VEGF receptor inhibitor group, and the ADM+ADM receptor inhibitor group, respectively. The expression levels of protein PI3K, Akt and eNOS was detected by Western blot.Results: Compared with the control group, the expression of PI3K/Akt and eNOS of the ADM group, the VEGF group, and the ADM+VEGF receptor inhibitor group were obviously increased. (As for the PI3K expression: ADM group/the control group t=-7.555 P=0.000<0.05, VEGF group/the control group t=-3.03 P= 0.007<0.05, ADM+VEGF receptor inhibitor group/the control group t=-3.832 P=0.001<0.05; as for Akt expression: the ADM group/control group t =-3.339 P=0.004<0.05, VEGF group/control group t=-3.805 P=0.001<0.05, ADM+VEGF receptor inhibitor group/control group t=-3.519 P=0.003<0.05; as for eNOS expression: the ADM group/control group t=-13.443 P=0.000<0.05, the VEGF group/control group t=-12.607 P=0.007<0.05, ADM+VEGF receptor inhibitor group/ control group t=-13.088 P=0.001<0.05).The expressions of PI3K/Akt and eNOS were further increased in a time-dependent manner. However, the expression of PI3K/Akt and eNOS in cells showed no difference between ADM+VEGF receptor inhibitor group and the ADM group (as for PI3K expression, ADM group: ADM+VEGF receptor inhibitor group 24h t=0.726 P=0.477>0.05, 48h t=0.199 P=0.844>0.05, 72h t=0.171 P=0.866>0.05. As for Akt expression, the ADM Group: ADM+VEGF receptor inhibitor group 24h t=0.501 P=0.624>0.05, 48h t=1.03 P=0.317>0.05, 72h t=0.946 P=0.357>0.05. As for eNOS expression, the ADM Group: ADM+VEGF receptor inhibitor group 24h t=-0.017 P=0.987>0.05, 48h t=0.430 P=0.672>0.05, 72h t=-0.370 P=0.715>0.05).The expression of PI3K/Akt and eNOS in ADM group were significantly increased than that in ADM+ADM receptor inhibitor group (As for PI3K expression: ADM Group/ADM+ADM receptor inhibitor group 24h t=3.456P=0.003<0.05, 48h t = 6.960 P=0.000<0.05, 72h t = 10.397 P=0.000<0.05; As for Akt expression, ADM Group/ ADM + ADM receptor inhibitor group 24h t=2.629P=0.017<0.05, 48h t=7.286 P=0.000<0.05,72h t=11.166 P=0.000<0.05; As for eNOS expression, 24h t= 4.685P=0.000<0.05, 48h t=6.960P=0.000<0.05, 72h t=19.297P=0.000<0.05). However, there is no difference in the expression of PI3K/Akt and eNOS in cells between ADM+ADM receptor inhibitor group and the control group (As for PI3K expression, ADM+ADM receptor inhibitor group/ control group 24h t=1.011P=0.325>0.05, 48h t=-0.335P=0.741>0.05, 72h t=0.031P=0.975>0.05; As for Akt expression, the ADM+ADM receptor inhibitor group/control group 24h t=-0.353 P=0.728>0.05, 48h t=-0.44 P=0.665>0.05, 72h t=-0.139P=0.891>0.05. As for eNOS expression, the ADM+ADM receptor inhibitor groups/ control group 24h t=-0.582P = 0.568>0.05, 48h t=-0.651P = 0.741>0.05, 72h t=-0.528 P=0.523>0.05).Conclusion: ADM and VEGF could activate PI3K/Akt signaling pathways and induce the expression of eNOS. VEGF receptor inhibitors couldn't interrupt the activation of PI3K/Akt signaling pathway and the expression of eNOS induced by ADM, indicating the activation of PI3K/Akt signaling pathway and the expression of eNOS induced by ADM not only through the indirect regulation of VEGF. ADM receptor inhibitor could effectively interrupt the activation of PI3K/Akt signaling pathway and induce the expression of eNOS.?ADM played a role in regulating tumor angiogenesis by activating the PI3K/Akt signaling pathway and inducing the expression of eNOS with the autocrine / paracrine mechanism.
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