The Effects Of Angiotensin-â…¡ And Angiotensin-â…¡ Type 1 Receptor On The Angiogenesis Of Papillary Thyroid Carcinoma

Papillary thyroid carcinoma (PTC) is the most common malignant thyroid tumor. Because of its unobvious clinical manifestation PTC are often diagnosed after the metastasis has occurred. Therefore, besides operation, to search for further therapies will be helpful to patients' prognosis.It has been well known that angiogenesis is closely correlated with the occurrence, development and metastasis of carcinomas. Angiotensin-â…¡(ANG-â…¡) is a main effector bioactive peptide in the renin-angiotensin system.Recently,studies have shown that ANG-â…¡is involved in the occurrence and development of tumor, ANG-â…¡may promote the growth and angiogenesis of tumor through stimulating ANG-â…¡type 1 receptor (AT₁R). Angiopoietin-2(Ang-2) was recently reported to play a critical role in pathologic angiogenesis, especially in interaction with the vascular growth factor (VEGF).There is no report about the expression of ANG-â…¡and AT₁R in PTC up to now . this study will investigate their effects on the angiogenesis of PTC through measuring the expression of VEGF, Ang-2, ANG-â…¡and AT₁R protein, and the microvascular density (MVD) marked with CD34 antibody by immunohistochemistry SP. It will offer theoretical basis for searching for new therapies on PTC through blocking the angiogenesis. Methods1 The expression of ANG-â…¡, AT₁R, as well as VEGF, Ang-2 protein were measured in 20 cases of PTC, 20 cases of benign thyroid neoplasm (including 10 cases of thyroid adenoma and 10 cases of nodular goiter) and 15 cases of normal thyroid tissue (which were got from the normal thyroid tissue adjacent to thyroid adenoma) , and the MVD marked with CD34 antibody by immunohistochemical SP technique.2 Evaluation of resultsImmunohistochemistry SP: The percentage of ANG-â…¡, AT₁R, Ang-2 and VEGF protein positive cells in five high-power fields(400x) in the section was estimated and graded into one of four categories. Immunostaining intensity for ANG-â…¡, AT₁R and Ang-2 protein was identified as follows: (-), <10%; (+), 10%～30%; (++), 31%～50%; (+++), >50%. For VEGF protein: (-), <5%; (+), 5%～25%; (++), 26%～50%; (+++), >50%.For the determination of MVD, a brown endothelial cell or a cell group was taken as a microvessel. The areas which comprised the most microvessels within a section were selected. Under a light microscope at 400-fold magnification, the microvessels were counted in three fields and the mean was calculated as the MVD value for each case.Two investigators independently evaluated the staining results. The average values were calculated.3 Statistical analysis: All the data were analyzed by SPSS10.0 statistical software package. The dosimetric data comparison by t test or by ANVOA; the comparison of counting data by x2 test and Fisher's exact test; correlation analysis by Spearman's rank correlation; the level of significant difference was a =0.05.Results1 The MVD was immunohistochemically stained with anti-CD34 antibody.The positive staining for CD34 was mainly localized in the cytoplasm of vascular endothelial cell. The MVD described as mean±standard deviation in PTC, benign thyroid neoplasm. and normal thyroid tissue were 57.60±13.11, 46.02±10.03 and 15.83±8.21, respectively. The data showed a significant diference among the groups (P <0.01).2 VEGF was mainly localized in the cytoplasm of thyroid follicle epithelial cells. It's staining pattern was major focal staining in PTC. The positive rate of VEGF in PTC, benign thyroid neoplasm and normal thyroid tissue were 85.00% (17/20 cases) ,55.00% (11/20 cases) and 13.33% (2/15 cases), respectively.The expression of VEGF was significantly higher in PTC than that in benign thyroid neoplasm and normal thyroid tissue (P <0.05 or P<0.01) ,respectively.The expression of VEGF in benign thyroid neoplasm was also significantly higher than that in normal thyroid tissue(P<0.05).3 The distribution of Ang-2 expression was similar to that of VEGF protein in PTC. Ang-2 protein is positive in 80.00% of PTC (16/20 cases ) , 45.00% of benign thyroid neoplasm (9/20 cases) and 13.33% of normal thyroid tissue (2/15 cases). the positive rate of Ang-2 in PTC was significantly higher than that in benign thyroid neoplasm and normal thyroid tissue(P<0.01 or P<0.05), respectively. The positive rate of Ang-2 in benign thyroid neoplasm was also significantly higher than that in normal thyroid tissue (P<0.05) .4 The diffusely expressed ANG-â…¡were observed in 75.00% (15/20 cases )in PTC. The positive rate of ANG-â…¡in benign thyroid neoplasm and normal thyroid tissue were 40.00% (8/20 cases) , 6.67% (1/15 cases) . The expression of ANG-â…¡was significantly up-regulated in PTC compared to benign thyroid neoplasm and normal thyroid tissue (P <0.05 or P<0.01) ,respectively.The expression of ANG-â…¡was significantly up-regulated in benign thyroid neoplasm compared to normal thyroid tissue(P<0.05).5 The distributing region of AT₁R positive cells was similar to that of ANG-â…¡in PTC. In PTC, benign thyroid neoplasm and normal thyroid tissue ,the rates of immuno-positive staining of AT₁R were 90.00% (18/20 cases),50.00% (10/20 cases)å’Œ6.67% (1/15 cases) , respectively. There were significant diferences among them (P <0.01).6 The expression of VEGF, ANG-â…¡and MVD values in the PTC group with lymph node metastasis were both significantly higher than that in the PTC group without lymph node metastasis (P < 0.05), no significant difference in the expression of Ang-2 and AT₁R between two group (P>0.05). On the other hand, the expression of VEGF, Ang-2, ANG-â…¡, AT₁R and MVD in PTC was not related to the patients' gender and Age. 7 The expression of ANG-â…¡was positively related with that of AT₁R, VEGF and MVD values in PTC (P<0.05 or P<0.01). The degree of AT₁R expression had significantly positive correlation with that of VEGF expression and MVD values in PTC (both P<0.05). Meanwhile, the VEGF protein expression also had a close relation with Ang-2 protein expression or MVD values (both P<0.05). But the expression of Ang-2 was not associated with that of AT₁R protein and MVD values (all P>0.05).Conclusion1 PTC growth depend on new angiogenesis,which play an important role in lymph node metastasis of PTC.2 There might be autocrine and paracrine of VEGF in PTC. The high expression of VEGF might promote the angiogenesis and metastasis of PTC. Ang-2 was mainly secreted by the cancer cells of PTC. Moreover, it might promote the angiogenesis of PTC in coordination with VEGF. But Ang-2 might not be an independent factor of the angiogenisis and metastasis in PTC.3 For the first time, the expression of ANG-â…¡and AT₁R in PTC were detected by immunohistochemical SP technique.The study suggested that ANG-â…¡and AT₁R was mainly secreted by the cells of PTC. There might be reciprocal induction and interaction between ANG-â…¡(ligand) and AT₁R(receptor), and there might be autocrine and paracrine of ANG-â…¡and AT₁R in PTC. Moreover,they may participate the angiogenisis and metastasis through promoting the growth of PTC. ANG-â…¡might up-regulate the expression of VEGF and Ang-2 protein and strengthen the activity of angiogenesis by stimulating AT₁R. The above results indicated that blocking the effects of ANG-â…¡might be a new strategy for the treatment of PTC.