Sildenafil Suppresses The Proliferation And Enhances The Apoptosis Of Hemangioma Endothelial Cells And Its Mechanism

BackgroundHemangiomas are the most common benign tumors in infants with the incidence rate of 10 to 12%,mostly are found at birth or shortly after birth.The majority of hemangiomas can be involuted spontaneously,but still 20%of them can not subside.According to the lesion's development process,hemangioma can be divided into proliferation period,regression period and completed regression period.Modern medicine advocates a positive early treatment attitude towards hemangioma.However,due to lack of sufficient understanding of its pathogenesis,resulting in unsatisfactory treatment effect overall.Treatment of hemangioma mainly include drug therapy,laser therapy,surgical therapy,cryotherapy,sclerotherapy and so on.Cryotherapy and sclerotherapy and laser therapy easily lead to hyperplasia or atrophic scars,pigmentation or hypopigmentation.Surgical resection is often used for partial hemangiomas.Drug therapy is an easy and effective way,it can contribute to early hemangioma regression and therapeutic purposes.The drugs for Hemangioma commonly are the following types:1.?-adrenergic blockers,with propranolol dominated,it is the first-line drug treatment of hemangiomas.2.Corticosteroids,with prednisone dominated.3.Others:a-interferon(IFN-a),imiquimod,antineoplastic agents cyclophosphamide(CTX),vincristine(VCR)and the like.The French physician Leaute-Labreze C and collegues accidentally discovered that propranolol can promote nasopharyngeal hemangioma retreat and published their findings in the"New England Journal of Medicine" in 2008.They started the precedent of using propranolol to treat infantile hemangioma.In recent years,propranolol has gradually replaced corticosteroids and become the first-line drugs.In 2012,Swetman et al reported an occasional regression of lymphatic malformation(LM)combining with pulmonary hypertension in three children following treatment with oral sildenafil,which served as an antagonist of phosphodiesterase isoform 5(PDE-5).This finding suggests that PDE-5 inhibitors represent a promising new drug which is possible to cure lymphatic malformations.However,The article did not provide lymphatic malformation diagnosis basis but show the photos of children lesions and MRI.All of the three children were not confirmed to be LM by surgery and pathology.According to the MRI manifestations,we think that the diagnosis of the children is more consistent with hemangioma combined with lymphatic malformations,previously dominated.Sildenafil is an inhibitors of phosphodiesterase type 5 and may function by inhibiting PDE-5.Inspired by these,tissue sections from patients with lymphatic,arteriovenous,venous malformations,and hemangioma tissues were selected for immunohistochemistry(IHC)to confirm the expression of PDE-5,based on the Swetman et al report.However,our study revealed that PDE-5 was expressed in the cytoplasm of endothelial cells in IH,not in the endothelia of LM.Therefore,we thought that sildenafil may affect hemangioma instead of lymphatic malformation.Previously,PDE-5 was implicated in the proliferation and apoptosis of pulmonary ECs and cancer cells.We assumed that sildenafil may promote regression of hemangiomas by regulating proliferation and apoptosis.To assess this hypothesis,the proliferation and apoptosis of specimen-derived hemangioma endothelial cells(HemECs)following treatment with sildenafil in vitro were studied and its potentially associated mechanisms were investigated.Purpose1.To detect PDE-5 expression in hemangioma and vascular malformation tissues.2.To investigate the proliferation and apoptosis of specimen-derived hemangiomaendothelial cells(HemECs)after sildenafil treatment.3.To study the potential mechanisms implicating with the inhibitor of differentiation 1(Id-1)expression.Methods1.Formalin-fixed and paraffin-embedded specimens of vascular anomalies for IHC were obtained randomly from the Department of Pathology of Qilu Hospital(Shandong University,Jinan,China)between 2000 and 2013.The specimens included lymphatic malformations(n=10),proliferating hemangiomas(n=8),involuting hemangiomas(n=2),arteriovenous malformation(n=10),venous malformation cases(n=10).The diagnosis of these anomalies was confirmed by patient medical histories,physical examinations,magnetic resonance imaging(MRI)and final pathological examinations.Lymphatic vessel endothelial hyaluronan receptor-1(LYVE-1),cluster of differentiation 34(CD34)and glucose transporter-1(GLUT-1)were used as markers for lymphatic endothelial cells,vascular endothelial cells and hemangioma tissues,respectively,to confirm the type of vascular anomalies.Subsequently,the expression of PDE-5 was assessed in the tissues of all the vascular anomalies.The present study was approved by Qilu Hospital Ethics Committee of Shandong University,and all the patients provided their consent.2.Specimen of proliferating hemangioma was obtained from patients by surgical resection and was confirmed by pathological examinations in the Department of Qilu Hospital,Shandong University.The tissue samples were rinsed with PBS and cut into smaller pieces.The majority of the pieces were used for culturing HemECs and the residual was maintained to estimate the expression of GLUT-1,CD 34 which served as the molecular marker in HemECs.The expression levels of CD34,von Willebrand factor(vWF)and PDE-5 were determined by immunocytochemistry(ICC).MTT assay were applied to study the proliferation and the viability of HemECs after increasing concentrations of sildenafil(0,1,3,5,10 and 15 ?M)for different durations(24,48 and 72 h)in vitro.HemECs were treated with increasing concentrations of sildenafil(0,1,5,10 and 15 pM)for 20 minutes before 50 ng/ml bFGF?50 ng/ml VEGF were added to continue incubating for 24 h.MTT assay were applied to study the suppressive effects of sildenafil on the proliferation of HemECs induced by bFGF and VEGF.The apoptosis of HemECs was determined following treatment with 0 and 5 ?M sildenafil for 24 h in vitro by flow cytometry.3.The HemECs were exposed to 0 and 5 ?M sildenafil for 24 h.The total RNA in the HemECs was extracted using TRI reagent and the Id-1 mRNA was reverse transcribed into cDNA using a PrimeScript RT-PCR kit.The cDNA was amplified by PCR using specific primers for Id-1.Differences in the threshold cycles between Id-1 genes and the reference gene(?-actin)were calculated.The value of the relative mRNA quantity for Id-1 genes were analyzed using the comparative Ct method(2-??Ct).The HemECs were exposed to 0 and ?M sildenafil for 24 h,and were collected for protein extraction using radio immunoprecipitation assay lysis buffer/phenylmethanesulfonyl fluoride.The protein samples were separated using a sodium dodecyl sulfate polyacrylamide gel electrophoresis gel kit.Immunopositive bands were examined using an enhanced chemiluminescence(ECL)detection system FluorChem Q and an ECL kit.The images were developed on X-ray film.A human TCA 8113 tongue squamous cell carcinoma cell line was used as a positive control for the protein expression of Id-1.Results1.Immunohistochemistry study show that CD34,LYVE-I and GLUT-1 were considered as positive markers for vascular endothelial cells,lymphatic malformation endothelial cells and hemangiomas endothelial cells,respectively.The expression levels of PDE5 were determined and demonstrated that the expression of PDE5 was present in the cytoplasm of endothelial cells of hemangiomas,but not in the lymphatic malformation and other vascular malformation endothelia.2.HemECs were identified using the markers vWF and CD34,and the positive expression of PDE-5 was determined.It was demonstrated that 5?M sildenafil exerted significant suppressive effects on the proliferation of HemECs following incubation for 24 h,compared with the control group(0 ?M sildenafil;t=3.220,P<0.01).This suppression gradually increased as the concentration increased in a dose-dependent manner.There was no significant suppression of the proliferation in HemECs treated between 24 h and 48 h(P>0.05).5 ?M sildenafil also exerted significant suppressive effects on the proliferation of HemECs induced by bFGF and VEGF(P<0.05).This suppression gradually increased as the time increased.However,an insignificant suppression of the proliferation was observed in HemECs treated with between 10 and 15 ?M sildenafil(P>0.05).Therefore,the present study considered 5?M and 24 h to be the minimal effective concentration and duration of sildenafil treatment for the suppression of HemECs.Following treatment with 5 ?M sildenafil for 24 h,the morphology of HemECs changed with the destruction of intercellular junctions and increasing particles in the cytoplasm under light microscopy.Results of the FITC-PI labeled flow cytometric analysis demonstrated a higher apoptosis rate at 10.5%compared with 2.53%in the control group.Therefore,5 ?M sildenafil significantly suppresses the proliferation and enhances the apoptosis in hemangioma endothelial cells in vitro.3.Following treatment with 5 ?M sildenafil for 24 h,the relative mRNA expression of Id-1 in the HemECs markedly decreased by 44.2%,according to the 2-??Ct method(t=9.749,df=4,P=0.0006<0.01).Additionally,the protein expression of Id-1 decreased following treatment with sildenafil,according to the grey scale of obtained immunoblotting bands.Conclusion1.PDE-5 is positive expressed in hemangioma.2.Sildenafil,which serves as an antagonist of PDE-5,had the property to suppress proliferation and enhance apoptosis in hemangioma endothelial cells in vitro.3.Both mRNA and protein expression levels of Id-1 in HemECs were down-regulated after sildenafil treatment.