Pathological And Cytological Study Of Invasion And Apoptosis In Human Intramuscular Vascular Malformation

PART ONE Evaluation of pathological morphology and Expression of matrix metalloproteinase-9 and Phospho-Tie2 in intramuscular vascular malformationObjective To indentify the character of IMVM pathology;to investigate the expression and significance of matrix metalloproteinase-9(MMP-9)and Phospho-Tie2 (P-tie2) in intramuscular vascular malformation(IMVM) and to analyze the relation between clinical manifestation and pathologic changes.Methods Specimens of 39 IMVMs and 10 normal craniofacial subcutaneous tissues were collected and stained with H.E and ABC Immunohistochemistry in order to observe the pathological change of the tissue morphology and to detect the expression of MMP-9 and P-tie2.Analysis program on image was adopted to compare the intensity of immunostaining in different samples.Results Observed under microscope,IMVMs were formed by various abnormal vessels which aggregated together in different sizes,developing within muscles. Accompanied by fat tissue,the endothelial cells arranged disorderly.The thickness of vessel walls varied.Both embolism and recirculation could be found in the lumen with growth of new capillaries.The expression levels of MMP-9 and Phospho-Tie2 were significantly higher in IMVMs than in normal subcutaneous tissues(P＜0.01). Their positive staining intensity had no statistically significant difference among the cases of different courses,locations,dimensions and infiltration degrees(P＞0.05).Conclusion The morphological abnormity of lumen connections and vascular cells formed the pathological foundation of IMVM.MMP-9 and P-tie2 may be involved in the pathogenesis and development of IMVM and assist the clinical and pathological diagnosis of IMVM. PART TWO Culture,identification and biological behavior study of endothelial cells from human IMVMObjective To explore the methods of isolation and cultivation of endothelial cells from IMVM(IMVM-EC);To study the characteristics configuration,phenotype, biological behavior on proliferation,migration,invasion and apoptosis of IMVM-EC; and to analyze its difference from human umbilical vein endothelial cell(HUVEC).Methods After IMVM specimens and umbilical cords were harvested sterilely, primary explant culure was adopted to isolate and purify IMVM-EC while collagenase digestion was used to culture HUVEC.The growth and morphology of two endothelial cells(ECs) were observed and compared under the inverted phase contrast microscope.FVⅢ-Ag,CD31,VEGFR,Phospho-VEGFR(P-VEGFR),PDGF, and Phospho-PDGFR(P-PDGFR) of cells were identified by immunofluorescence and immunochemistry.Growth curves of IMVM-EC were scaled by MTT assay.ECs were cultured on rat tail collagen to investigate the ability of tube formation.Scratch assay and Transwell test were performed to compare the migration and invasion of two ECs.Cell apoptosis were measured by flow cytometer under the condition of normal culture or FBS starvation.Results After 3-5 days,ECs could be seen from the margin of primary explants of IMVM.During 3 weeks of primary culturing,IMVM-ECs proliferated and filled the 25cm~2 culture bottle,showing cobblestone monolayer.After passage,IMVM-ECs entered the log phase of the proliferation in 3-6 days and partly represented the shape of tube formations,then aged in 4 passages.Both IMVM-ECs and HUVECs displayed the positive staining of FVⅢ-Ag and CD31.VEGFR,P-VEGFR,PDGFR,P-PDGFR were only slightly positive stained in HUVECs,but not in IMVM-ECs. IMVM-ECs and HUVECs had the similar ability of tube formation in vitro. Compared with HUVECs,IMVM-ECs indicated more powerful capability of migration and invasion,and more resistance to apoptosis either in normal culture condition or in starvation culture.Conclusion The model of endothelial cell culture from IMVM can be established successfully by using primary explant culture.The key of cultivation is adhesion of endothelium side of explant to the surface of culture bottle,purification of ECs by scraping the useless cells,proliferation of ECs by adding growth factors to the medium.In contrast with HUVECs,IMVM-ECs displayed biological character including lack of some cell surface antigens,enhancement of capacity in tube formation,migration,invasion and resistance to apoptosis.The defects and abnormal behavior of IMVM-EC may accelerate the aggravation of IMVM.PART THREE Molecular mechanism of invasion and apoptosis in Intramuscular vascular malformation1.Gene and protein expression of MMP-9 and P-tie2 in IMVMObjective To investigate the expression of MMP-9,P-tie2(Tyr992) in IMVM tissues from both levels of gene transcription and protein translation and to identify their relation with IMVM pathological development.Methods Specimens of IMVMs and normal craniofacial subcutaneous tissues were collected.RT-PCR and Western Blot were performed to detect the mRNA and protein expression of MMP-9 and P-tie2 in samples.Expressions ofβ-actin in each specimen were defined as inner controls.The intensities of different expressions were analyzed statistically.Results The mRNA levels of MMP-9 and P-Tie2 were expressed more highly in IMVM specimens(n=17) than in the comparison group(n=7)(P＜0.05).There were no statistically difference of the mRNA expression in different locations and therapy times.13 of 17 IMVMs' samples showed protein expression of P-tie2 and 15 of 17 showed expression of MMP-9,only 1 of 9 samples of nomal tissues did.The positive ratios were different(P＜0.05).Conclusions MMP-9 and P-tie2 are involved in the pathological development of IMVM.2.Study of MMP-9,P-tie2 expression and the related signaling pathway of PI3k/Akt in IMVM-ECObjective To investigate the expression and change of MMP-9,P-tie2,PI3K/Akt signaling pathway in IMVM-ECs from both levels of gene transcription and protein translation and to explore the pathological molecular mechanism of IMVM in invasion and apoptosis.Methods On the basis of culture methods of IMVM-EC,the vascular smooth muscle cells(VMSC) from IMVM were isolated and purified by primary explant culturing.Cells were divided into 3 groups:IMVM-EC,HUVEC and VSMC.ECs were induced to apoptosis by starvation culture.RT-PCR was used to detect the mRNA expression of MMP-9 and P-tie2 in different ECs.ECs were treated with LY294002 and SU11248.The expression changes of MMP-9,P-tie2,Akt and P-Akt protein were analyzed by Western Blot.Results The mRNA levels of MMP-9 and P-Tie2 were expressed more highly in IMVM-ECs than in HUVECs.VSMCs from IMVM didn't show the positive protein expression of MMP-9 and P-tie2.The IMVM-ECs under normal and apoptosis condition indicated the expression of P-Akt,while HUVECs didn't.LY294002 was able to prevent the self-phosphorylation of Akt,but didn't interrupt the expression of P-tie2 in IMVM-ECs.SU11248 could suppress the expression of P-tie2 located in the surface of two ECs and induced their death.Conclusions Elevated expression of P-Akt(Ser473) may be involved in the pathological behavior of IMVM-ECs.P-tie2 may activate the signaling pathway of PI3k/Akt to improve IMVM-ECs' resistance to apoptosis.IMVM-ECs could express high level of MMP-9 to enhance the invasive capability.Specific inhibitors of MMP-9 and tyrosine kinase on IMVM-ECs are potential to become the selective biological therapy of IMVM.3.Immunohistochemical study of P-Akt in IMVMObjective To evaluate the expression and significance of Phospho-Akt(P-Akt, Ser473) in IMVM and identify its relation with IMVM's pathological development.Methods 39 clinical specimens of IMVM and normal craniofacial subcutaneous tissues were collected and stained with ABC Immunohistochernistry.The positive staining was calculated and analyzed.Results 15 specimens of 39 IMVM tissues were stained with P-Akt,but the normal tissues(n=10) didn't show the staining.The positive ratios were different(P＜0.05)Conclusions P-Akt may be involved in the pathological development of IMVM.