Experimental Study On Endothelial Cells Apoptosis Of Infantile Hemangiomas Induced By Fas Ligand

Hemangioma is the most common tumor of infancy and childhood,occuring in 4%to 12%of white infants.These lesions are three to five times more common in girls than in boys,there is an increased frequency of hemangiomas in premature infants,with a reported incidence of 23%in neonates who weigh less than 1200g.Hemangioma occur most often in the craniofacial region(60%),followed by the trunk(25%) and extremities(15%),little is understood about the pathogenesis of infantile hemangiomas to date.Several studies have revealed that expression of proliferating cell nuclear antigen(PCNA),erythrocyte-type glucose transporter protein- 1(GLUT- 1) and lymphatic endothelial hyaluronan receptor- 1(LYVE- 1) as well as CD 133,an important marker for endothelial progenitor cells(EPCs) in endothelial cell of infantile hemangioma,which indicated the endothelial cells in infantile hemangioma are arrested in an early developmental stage of vascular differentiation.The immature,incompletely differentiated immunophenotype of proliferating infantile hemangioma may contribute to their rapid growth.Many studies suggested that programmed to death have a important role in hemangioma,a lot of regulatory proteins of apoptosis have been found to express in infantile hemangioma,such as bcl-2,bax,bad,bak,C-fos.In our previous study,we found the number of Fas(+) cells can be observed to increased from the middle proliferating stage to the late proliferating stage of infantile hamangioma,similar variance have been found in the number cells of T cell of infantile hernangioma,which expressed FasL antigen.We make the hypothesis that the apoptosis of endothelial cell in IH may be caused mainly by T cells expressing FasL protein,aiming at the Fas antigen of IH cells and mediated by Fas-FasL pathway.Part one Culture and identification of endothelial cells from IH and observation of biological character in vitroObjective:To explore the method of cultivation and purification the endothelial cells from infantile hemangiomas in vitro,immunohistochemical stain was performed to identificate the cell cultured form IH,the purity of endothelial cell was detected by flow cytometry analysis,the biological character of the cultured cell has been observed in vitro.Method:Small fragments of fresh operative specimens were obtained from 14 infantile hemangiomas that came form Shanghai Changhai hospital,Second Military Medical University and Shanghai Xinhua hospital,Shanghai Jiaotong University.Pieces of fresh operative hemangiomas tissue were submerged in D-hank's medium and stored at 4℃,then transport it from operative room to cell culture room.The endothelial cells of IH were cultured by the explants of specimens.The explants was removed from the dishes after the endothelial cell grew out.The cell colonies,which did not have the gross morphology of endothelial cells(under inverted phase contrast microscopy),were detached by scraping and removed from the dish by aspiration.The colonies that exhibited the characteristic cobblestone morphology of endothelial cells were allowed to proliferate in the dish for 1 week,selected as before.After 3-4 weeks culture,the primary culture cell grew the major of dish.Immunohistochemical staining of anti-CD31,anti-CD34 and anti-factorⅧassociated protein was performed respectively to identificate the EC origin of isolated cells.The purity of endothelial cell was detected with FITC-CD34 by flow cytometry analysis.The number of cultured cells was counted with micro grid technique and growth curve of cell was drawled.Results:In all 14 explants,endothelial cells of IH from 8 specimens grew in tissue culture.The morphology of cultured endothelial cells cold be divided into 2 types:the round cell with well defined,centrally located nuclei and distinct cell borders,the shape cell,which exhibited cobblestone morphology after confluence.Cultured EC were homogenously positive for factorⅧassociated protein,CD31,CD34 immunohistochemical staining,indicated the EC origin.Formation tube was found in endothelial cell of IH.The number of CD34(+) cell was 75.27%±3.73%in endothelial cell by flow cytometry analysis.Grow curve based on microgrid technique indicated EC began to proliferated rapidly after 7 days of plating,the number of EC was maximum at 11 to 12 days of plating,follow this,the number of EC began to plateau in grow for 3 days,then decreased slowly.The primary cell can be subculture for no more than 3 erasConclusion:The EC of IH can be isolated and cultured successfully by modified explant method,verified the endothelial origin by immunohistochemical staining,the purity of EC is high in the subculture cell,and Formation tube was found in endothelial cell of IH. Part two Expression of Fas and Fas ligand on endothelial cell of IHObjective:The flow cytometry analysis and fluorescencere quantitative reverse-transcriptase polymerase chain reaction(RT-PCR) assay were used to detect the expression of Fas and Fas ligand on endothelial cell of IH.Method:the EC of IH and umbilical vein were subculfivated and plated on plastic dish,after 1 week of culture,the cells were digested with 0.25%trypsin solution and centrifuged(1000r/min,5 min),After harvesting,the cells were detected the Fas and FasL antigen of expression on EC of IH and umbilical vein by FACS,fluorescencere quantitative PCR was performed on cell to detect Fas and FasLmRNA expression levels. The results were compared with that of Jurkat cells.Fas primers were 5'- TTGCTAGATTATCGTCCAAAAGTGT-3'and 5'- GCACTTGGTGTTGCTGGTGAGT-3',amplification product was 205 bp;FasL primers were 5'-TTCAGCTCTTCCACCTACAGAAGGA-3' and 5'-TCACTCCAGAAAGCAGGACAATTC-3',amplification product was 219 bp,β-actin primers were 5'-ACCACAGTCCATGCCATCAC-3' and 5'-TCCACCACCCTGTTGCTGTA-3',amplification product was 450 bp,amplification was performed under the following conditions:95℃3 min,{95℃20s61℃20s72℃30s}×50cycles,72℃5rain,55℃—95℃(melt curve).Results:FCM detection showed that Fas expression rate of IH was 90.97%±2.36% and FasL expression rate was 4.17%±1.75%,Fas expression rate of HUVEC was 25.07%±7.60%and FasL expression rate was 4.45%±40.50%;results of RT-PCR showed that the level of Fas mRNA of IHEC was 1.260±0.721 and FasLmRNA was 0.038±0.022, the level of Fas mRNA of HUVEC was 0.354±0.170 and FasLmRNA was 0.022±0.011. The level of Fas mRNA of IHEC compared with that of Jurkat cells 1.448±0.059,there were no significant difference between IHEC and Jurkat cells.Conclusion:The expression of Fas antigen on IHEC is high,but that of FasL antigen on IHEC is low. Part three IHEC apoptosis induced by soluble Fas ligandObjective:To investigate the effect of IHEC apoptosis induced by different doses of sFasL,To examined the susceptivity of apoptosis on IHEC,HUVEC line and Jurkat cell line induced by sFasL,as well as the effect of FasL neutralizing antibody NOK-2 block the apoptosis induced by sFasL.Western Blot analysis was carried out to determine the protein of active Caspase 3,8,9 after IHEC apoptosis induced by sFasL.Method:5 ng/ml,20 ng/ml,100 ng/ml doses of sFasL were added to EC medium respectively,all of the above cells were cultured for 6h,18h and 36h,then IHECs were collected carefully and stained with PI and Annexin V-FITC conjugate,apoptosis of IHECs was measured by FACScan.Apoptosis of normal grown IHECs without sFasL were negative control group;IHECs and HUVECs were incubated with sFasL at 100ng/ml,in another group IHECs and HUVECs were incubated with sFasL at 100ng/ml and neutralizing FasL monoclonal antibody NOK-2(10ng/μl),Jurkat cells were incubated with sFasL at 5ng/ml,in another group Jurkat cells were incubated with sFasL at 5ng/ml and neutralizing NOK-2(10ng/μl),all of the above cells were cultured for 18h,then,apoptosis of IHECs,HUVECs and Jurkat cells were measured by FACScan.Apoptosis of normal grown IHECs,HUVECs and Jurkat cells were negative control group respectively.Western Blot analysis was performed to detect the protein of active Caspase 3,8,9 after IHEC apoptosis had been induced by sFasL for 18h.The protein of active Caspase 3,8,9 of norm grown IHECs was negative control group.Results:Results shows:(1)The total apoptotic rate of IHECs were 9.92%±0.84%; 10.79%±2.01%;10.33%±0.98%after incubation with 5 ng/ml sFasL for 6h,18h,36h; The total apoptotic rate of IHECs were 11.03%±1.62%;11.36%±1.13%;11.46%±1.67% after incubation with 20 ng/ml sFasL for 6h,18h,36h;the total apoptotic rate of IHECs were 11.68%±1.63%;14.86%±0.40%;16.40%±1.08%after incubation with 100 ng/ml sFasL for 6h,18h,36h。(2) The apoptotic rate of IHECs were 18.12%±0.24 %;12.85±0.93%after incubation with 100 ng/ml sFasL and 100ng/ml sFasL+NOK-2 (10ng/μl) for 18h,the difference were significant(p＜0.01);The apoptotic rate of HUVECs were 14.53%±1.49%,11.17%±1.19%after incubation with 100 ng/ml sFasL and 100ng/ml sFasL+ 10ng/μl NOK-2 for 18h,the difference were significant(p＜0.01); The apoptoticrate of Jurkat cells were 61.59%±11.37%;16.59%±4.66%after incubation with 5 ng/ml sFasL and 5ng/ml sFasL+ 10ng/μl NOK-2 for 18h,the difference were significant(p＜0.01)。(3) the apoptotic rate of IHECs induced by sFasL that was 18.12%±0.24%was higher than that of HUVECs 15.87%±0.48%(p＜0.01),and was lower than that of Jurkat cells 61.59%±11.37%(p＜0.01 ).(4) After IHEC had been induced by 100ng/ml sFasL for 18h,Western Blot showed that the fragment of Caspase 8 were 43kDa and 18kDa,which relative gray scale to that ofβ-actin were 0.5,0.08,compare with the control group0.09,0,the difference were significant(p＜0.01);The fragment of Caspase 3 were 19kDa and 17kDa,which relative gray scale to that ofβ-actin were 0.26, 0.16,the control group did not express those fragments;The fragment of Caspase9 were 37kDa,35kDa and 17kDa,which relative gray scale to that ofβ-actin were 0.43,0.04 and 0.07,compare with the control group0.44,0.04,0.07,the difference were no significant.Conclusion:IHEC apoptosis can be induced by sFasL,the viability of apoptosis is in dose-dependant manner,no correlate with time.The susceptibility to sFasL among Jurkat cells,IHECs and HUVEC is vary,Jurkat cells was highest,lower was IHECs and lowest was HUVECs.The apoptotic effect of sFasL on cells can be blocked by NOK-2,IHECs apoptosis had been induced by sFasL,Western Blot detect the expression of active Caspase 8,3,but not Caspase 9,indicated that the apoptosis of IHEC is by Fas/FasL pathway.Part four Apoptosis of infantile hemangioma endothelial cells induced by H9 cells lineObjective:To detect the expression of FasL on H9 cells line after treated with IL-12 and exminated the apoptosis of IHECs co-cultured with H9 cells after treated with IL-12.Method:(1) H9 cells line were treated with 25ng/μl IL-12 for 12h,then H9 cells were collected and to detect the FasL expression by FCAScan,as well as the FasLmRNA expression by RT-PCR,H9 cells untreated with IL-12 were negative control group.(2) IHECs were subjected to co-culture with H9 cells for 18h which prior to treat with IL-12 (25ng/μl) for 12h;IHECs were co-cultured with H9 cells which prior to treat with IL-12(25ng/μl) for 12h then were neutralizing NOK-2 for 1h.The apoptosis of IHECs was detected by FACScan,the negative control group was IHECs co-cultured with H9 cells untreated with IL- 12.Results:(1) FACS showed the FasL expression rate on H9 cells treated with IL-12 was 46.65%±3.26%,was higher than that of untreated group 4.51%±0.85%,the difference was significant(p＜0.01 ).RT-PCR results showed the FasLmRAN expression rate on H9 cells treated with IL-12 was 0.6±0.32,was higher than that of untreated group 0.097±0.01, the difference was significant(p＜0.01).(2)The apoptosis of IHECs co-cultured with H9 cells treated with IL-12 was 26.02%±41.70%,which was higher than that of IHECs co-cultured with H9 cells untreated with IL-12 13.69%±1.68%(p＜0.01 ),it also was higher than that oflHECs co-cultured with H9 cells neutralized with NOK-2(p＜0.01 ).Conclusion:H9 cells line stimulated with IL-12 can express FasL.The apoptosis of IHECs can induced by H9 cells treated with IL-12 and the apoptotic effect of H9 cells expressed FasL can be blocked by sFasL neutralizing antibody NOK-2.