The Effects Of Vascular Endothelial Growth Factor, Angiopoietin-2, Angiotensin-Ⅱ And Angiotensin-Ⅱ Type 1 Receptor In The Angiogenesis Of Patients With Graves' Disease

ObjectiveGraves' disease (GD) is the most common autoimmune thyroid diseases, which is characterized by diffuse goiter, thyrotoxicosis, and ophthalmopathy. Up to now there are not exact pathogenetic factors of its development, so it is impossible to treat the basic in Graves' disease yet. The classic therapies for GD are anti-thyroid drugs (AID), radioiodine (¹³¹1) and surgical subtotal, which have different advantages and disadvantages respectively. Interventional medical technique presented us a new method in treating GD. There is not long-term experience and systemic research yet, but it presents a new clue for the treatment. It has been proved, through not only pathology but also ultrasound, that there are conspicuous angiogenesis in patients' thyroid with Graves' disease. So it is important to study the mechanism of the angiogenesis in the GD patients.Angiopoietin-2(Ang-2) was recently reported to play a critical role in pathologic angiogenesis, especially in interaction with the vascular growth factor (VEGF).Angiotensin- II (ANG- II) is a main effector bioactive peptide in the renin-angiotensin system, acting not only as a vasoconstrictor but also as a growth-promoting and angiogenic factor via type 1 angiotensin II receptors (AT₁R). There were many results suggested that ANG- II may stimulate the production of VEGF and Ang-2 via AT₁R. There are few reports about the role of VEGF and no report about the role of Ang-2, ANG- II, AT₁R in the GD patients up to now. To investigate the role of them in angiogenesis of Graves' disease, this study will measure the expression of VEGF, Ang-2, ANG-II and AT₁R protein and the microvascular density (MVD) marked with CD34 by S-P immunohistochemistry .Methods1. 35 cases of human Graves' disease thyroid tissues and 11 cases of normal thyroid tissues (around innocent thyroid adenoma) were obtained. The expression of VEGF, Ang-2, ANG-II, AT₁R protein were detected and MVD marked with CD34 antibody was measured by S-P immunohistochemical method.2. Evaluation of resultsAll slides were evaluated without knowledge of patient identity or clinical status by two pathologists. The average values were calculated.The determination of VEGF, Ang-2, ANG-II, AT₁R protein positive results according to the Mattern' method. The cells containing brown particles were taken as positive cells while the background was clear. The percentage of ANG- II, AT₁R, Ang-2 and VEGF protein positive cells under five high-power fields(400X) was classified into four groups: (0 point), <10%; (1 point),≥10%～<30%; (2 points),≥30%～<50%; (3 points ),≥50%.The intensity of immunostaining was classified into four groups also: (0 point), negative; (1 point), weak; (2 points), mild; (3 points), strong. Sum of them was the last resuls: (-), (0 point); (+), (1～2 points); (++), (3～4 points) ; (+++), (5～6 points).For the determination of MVD value according to the Weidner' method, a brown endothelial cell or 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 X magnification, the microvessels were counted in three fields and the mean was calculated as the MVD value for each case.3. All the data were analyzed by SPSS10.0 statistical software package. The correlation analysis by Spearman's rank correlation, the comparison of counting data by x² test, the dosimetric data comparison of two groups by Student t test, the level of significant difference wasα=0.05.Results1. Immunohistochemistry SP Positive CD34 was localized in the vascular endothelial cells. VEGF, Ang-2, ANG-II and AT₁R protein was brown particles mainly localized in the cytoplasm and membrane of cells, a few nuclei were positive in AT₁R protein. Expression of VEGF, Ang-2, ANG- II, AT₁R and CD34 protein were enhanced in the areas where thyroid epithelial cells were over-hyperplastic and lymphocytes greatly infiltrated.2. Expression of VEGF, Ang-2, ANG- II, AT₁R protein and MVD value of Graves' disease thyroid were significantly higher than those of normal thyroid tissues(P<0.05).3. A significant correlation between VEGF, Ang-2, ANG- II, AT₁R protein expression and MVD was found in our research (VEGF r =0.723; Ang-2 r =0.675; ANG-II r =0.529; AT₁R r=0.747; all P<0.05).4. VEGF expression was significantly associated with that of Ang-2(r= 0.351, P<0.05); VEGF expression was significantly associated with that of ANG- II ( r= 0.508, P<0.01); VEGF expression was significantly associated with that of AT₁R( r= 0.537, P<0.01); ANG- II expression was significantly associated with that of AT₁R ( r= 0.423, P<0.05); ANG-II expression was significantly associated with that of Ang-2 ( r=0.387, P<0.05); Ang-2 expression was significantly associated with that of AT₁R(r= 0.493, P<0.01).Conclusions1. For the first time, this study found that expression of Ang-2, ANG- II, AT₁R protein in thyroid tissues with Graves' disease are significantly higher than those in normal thyroid tissues. Expression of VEGF, Ang-2, ANG- II and AT₁R protein are all positively correlated with MVD in Graves' disease. These results suggest that VEGF, Ang-2, ANG- II and AT₁R protein may play critical roles in angiogenesis of Graves' disease. 2. The expression of VEGF, Ang-2, ANG- II and AT₁R protein is positively correlated with each other . These results suggest that they may play a synergistic effect in angiogenesis of Graves' disease. ANG- II may stimulate the production of VEGF and Ang-2 via AT₁R.The above results suggest that it may be a new strategy to treat Greaves' disease through blocking the effects of VEGF, Ang-2 , ANG-II and AT₁R.