Isolation,Purification And Characterization Of Endothelial Cells From Human Brain Arteriovenous Malformation

Objectives: Cerebral arteriovenous malformation(cAVM)is commonly congenital vascular abnormalities,which is consisted of abnormal arteries,veins and arterialized veins.In the early phase of placental development,primordial arteries and veins are directly connected without the interposition of capillaries.Due to the dysplasia of capillaries,the connections of arteries and veins are remained.Intracranial hemorrhage is the most common clinical presentation of cAVM,resulting in a high risk of death and disability.Currently,many related studies show that the mechanism of cAVM is related to artery pressure disorder,vessel malformation,high blood flow rate and steal phenomenon.The disorder of blood vessels exposed in the high fliud shear stress,which will induce the activation of molecular pathways in the smooth muscle cells(SMCs)and the endothelial cells(ECs).Which is induced the proliferation of related cells and the remodeling of vascular.Many animal studies have demonstrated that the pathological of cAVM include heterogeneously thickened vessel walls,splitting of the elastic lamina,thickened endothelial layers,loss of endothelial continuity,lack of tight and adherent junctions and filopodia directed into the lumen.ECs play an important role in cAVM formation.To find a more effective and more convenient way to isolate and culture ECs is the basic study of cAVM formation mechanism.Methods: Ten patients with cAVM were identified by the magnetic resonance imaging(MRI),digital subtraction angiography(DSA)and pathology.However the superficial temporal vein(STV)of three epilepsy patients set as control group.All the samples above were collected from the surgical treatment of the patients in Beijing Tiantan Hospital.The immunoflurescence of vessel tissues were performed with von Willebrand Factor(vWF)and ?-smooth muscle action(?-SMA).Then,we collected the remaining samples for isolation and culture of ECs.The further isolated cells were selected by flow cytometry to obtain more purity ECs.The morphological changes of selected ECs were observed by light microscopy.Then the purity of ECs was identified by flow cytometry.The immunocytochemistry of selected cells were performed with CD31,CD144,vWF and ?-SMA.The Dil-AcLDL phagocytic was also performed to show the selected cells with ECs function.The changes of ECs biological characteristics were detected by cell migration,invasion and cell tubule formation assay,which were used to analyze and compare with the difference between cAVM and STV groups.Results:1 Clinical informations of cAVM and STV pateintsIn this study,ten patients of cAVM and three STV patients were analyzed.In the cAVM group,the clinical symptoms were excessively serious and were characterized by hemorrhage(7 cases)and epilepsy(3 cases).The size of cAVM nidus was between 40 to 70 mm.Each patient underwent surgical treatments.Seven patients of cAVM group were successfully isolated and cultured.The images of cAVM were shown in MRI and DSA.2 Immunofluorescence staining of vWF and ?-SMA in tissuesThe expressions of vWF and ?-SMA were measured by immunofluorescence staining in both STV and cAVM groups.The vWF was specifically expressed in ECs,and the ?-SMA localized in vascular smooth cells.3 The selection of primary isolated cells by flow cytometryUse the flow cytometry would separate more purity ECs.After cell selection,which both express CD31 and CD144 were cultured again.4 Histopathological changes and cell property of selected cellsUnder observation with microscope,isolated cells grew initially in separate colonies with round borders and cobblestone appearance.Used flow cytometry to identify isolated cells shown that CD31 and CD144 positive cells were 90.60% in cAVM group and 98.4% in STV group.5 Immunofluorescence staining of CD31,CD144,vWF and ?-SMA in cultured cellsBy the immunofluorescence staining,we obtained the expression levels of CD31,CD144,vWF and ?-SMA in cultured cells in cAVM and STV groups.In both groups,the expressions of above indicators were observed.6 Dil-AcLDL phagocytic test of ECsThe results of Dil-AcLDL phagocytic test indicated that the isolated cells had the ability of Dil-AcLDL ingestion in each group,which identified the isolated cell to ECs.7 Tubule Formation Assay of ECsCompared to the STV control group,the ability of tubule formation were dramatically decreased in the cAVM group(P < 0.05,n = 3).The total length of tubule in cAVM group was 12.58±0.30 mm/mm2,while the total length in the STV group was 22.42±0.86 mm/mm2.8 The migration and invasion of ECsCompared with STV group,the abilities of cell migration and invasion were remarkably decreased in the cAVM group(P < 0.05).The numbers of migrated cells in cAVM group were 596.50±29.07.While,the cell numbers in STV group were 28.42±8.56.In the invasion test,the numbers of incasion cells in cAVM and STV groups were 375.56±24.49 and 31.50±3.76,respectively.Conclusions: It was an effective method to get a mass of ECs in vitro by collagense digestion and fluorescent cell sorting.Our results indicated that the migration,invasion and tubule formation ability of ECs in cAVM group were dramatically decreased,which were closely associated with the formation mechanism of cAVM.