Study Of Clinical Pathology And Biology Of Vascular Smooth Muscle Cells Of Cerebral Arteriovenous Malformations

PART IHistopathology and immunochemistry study of cerebral arteriovenous malformations after intravascular embolization treatment Objective: To study the pathological change of cerebral arteriovenous malformations after intravascular embolization treatment Method: 11 specimens resected from the patients who had the history of preoperative embolization were analyzed by HE and immunohistochemisty assay. The antibody for immunochemistry were Proliferative nuclear cell antigen (PCNA),Osteopontin(OPN),Smooth Muscle Actin(SMA) and Vascular epithelial growth factor (VEGF).Result: There were inflammatory reactions varying in degree to embolic materials, such as lymphatic cell infiltration in the blood vessel wall , intimal hyperplasia, and accumulation of foreign body giant cells(FBG) . In some vessels ,recannalizaiton in the embolized vessels could be observed. The result of immunoassay demonstrated that Vascular smooth muscle cells attended in the neointimal formations . OPN can be found in the neointimal and FBG. Some capillary -like microvessels appeared in the tissue adjacent to nidus ,with high percentage of positive immunostain for PCNA and VEGF in endothelial cells, indicating active neoangiogenesis.The Index of PCNA positive endothelial cells in preoperative embolized patients (19. 62±8. 43) was significantly higher, comparing with that of 12 hemorrhagic CAVM patients(10.13 ± 7.21) (P<0.05) .Conclusion: Embolic materials can cause inflammatory reactions .moderatereactions were helpful in the formation of permanent emboli. Recannalization and sign of perinidal neovasularization sometimes could be found. PART IIHistopathology and immunochemistry study of cerebral arteriovenous malformations after stereostactic Gamma Knife Radiosurgery Objective: Stereotactic Gamma Knife Radiosurgery (GKRS) is a major choice for the treatment of Cerebral ArteriovenousMalformations(CAVM).This research focused on the pathological changes and their significance after GKRS and the proliferation and phenotype of VSMC in the pathophysiological process related to radiotherapy .. Method : Five surgical specimens were analyzed by HE stain and immunohistochemistry assay The monoclonal antibody PCNA ,SMA,OPN and VEGF were used for immunochemistry stain . One specimen were checked with electromicrscope.Result: Progressive angiopathological changes could be observed. Signs of endothelium damage appeared in the early stage , followed by proliferation of VSMC and expansion of extracellular matrix in the medium, which cause progressive thickening of the wall and obliteration of the lumen. Progressive degeneration of the wall and total hyalinization were the signs of the end-stage. Recannalizations could be observed .OPN expression and PCNA stain were positive in the walls which showed sign of early-stage damage. Electromicroscopy examination showed sign of damage in endothelial cell and VSMC and significant deposition of collagen in the intimal .medium and adventia layers.Conclusion : Proliferation , migration and degeneration of VSMC might be the key factor in the progressive angio — pathological process in the pathophysiology of CAVM treated by GKRS.PART IIIExpression of Osteopontin and its significance in cerebral arteriovenous malformationsObjective: As a marker gene of synthetic vascular smooth muscles and major chemotactic agent for major cellular component of blood vessels, Osteopontin(OPN) is considered to play an important role in several kinds of pathological vascular remodeling process . This research was intended to analyze the expression and its significance in Cerebral Arteriovenous Malformations and the pathological changes related to Gamma Knife stereo tactic Radiosurgery (GKRS) and intravascular embolization treatment. Method: The 42 specimens were divided into three groups: 5 in the group of having history of GKRS, 11 in the group of having history of embolizations treatment ,the other 26 without any preoperative interference were in the other one The expression of OPN were studied respectively. Result: OPN expression could be detected in 22 of 26 specimens without any preoperative treatment . The expression was mainly in the venous part of CAVM, especially in the tortuous vein with irregularly thickened wall . In the arterial part ,the expression could sometimes be find in the atherosclerotic-like lesions There is no statistically significant difference in the extent of OPN expression between groups with or without history of hemorrhage ,or between those groups divided according to their nidus size .In the group with history GKTRS, OPN expression can be find in arteries with early stage radio-effect in two patients; in the group with history of embolization , the intravascular expression of OPN was concurrent with the appearance of neointima and foreign body giant cell.Conclusion: OPN expression can be found in the CAVM vessels, which means active vascular remodeling of CAVM, and the expression in those with history of GKRS and embolization treatment suggest it may play an important role in the pathophysiolgic process related to the treatments ;PART IVCulture and identification of vascular smooth muscle cells from human arteriovenous malformationsObjective: To developed a procedure for the isolation and culture of vascular smooth muscle cells from human cerebral arteriovenous malformationMethod: The surgical specimens were carefully dissected ,digested with collagenase type IV . The cell population then was seeded on the dishes percoated with gelatin for primary culture. The cell line derived was then analyzed by cellular morphology ,immunochemistry and electromicroscopy assayResult : The first growing cell colony appeared 6 to 8 days after initial seeding. The growing cell clone showed characteristics of "hill-and-valley" growth pattern. In immunochemistry assay , more than 95% cells were positive for SMA stain , while negative for CD34. Myofilament and dense plaque could be observed in electromicroscopy The morphological ,immunochemical and ultracstructural characteristics of cultured cells were consistent with that of VSMC, indicating the harvested cell were purified VSMCs. The cells still grows well after 18 generations.Conclusion: A purified VSMC cell line can be developed by collagenase digest assay and the cells can be used as a cellular model in vivo.PARTVThe proliferation and migration of VSMC cultured from human arteriovenous malformations under different condition in vitro Objective: To study the effect of basic Fibroblast Growth Factor (bFGF) on cellular proliferation and Osteopontin(OPN) on the migration of VSMC cultured from human cerebral arteriovenous malformations .Method: Cell counting , MTT assay were utilized to analyze the stimulative ability of bFGF on cellular proliferation , RT-PCR and Western blot to analyze its effect on mRNA and protein expression of OPN. The cellular migration model was used to assess the effect of OPN on VSMC migration . Result: Cell counting analysis and MTT assay showed that bFGF cansignificantly stimulate the proliferation of cultured VSMC, In RT-PCR assay, The level of OPN/β -actin in bFGF treated group and control group were 0.39 ± 0.069 and 0.24 ± 0.049 respectively .there was statistical difference (P<0.05) The OD of OPN band showed in western blot were 267.91 ±22.16 and 171.735±29.77 in the experiment and control group .which also showed statistical difference (P<0.05) . The distance of cellular migration were 298.6 ±23.92 um and 164.1 ±20.1 um in OPN and control group respectively , the difference were statistically significant (P<0.01).Conclusion: Osteopontin can significantly enhance the ability of cellular migration .and bFGF can stimulate cellular proliferation and expression of OPN.