Effect Of Timolol Combined With Propranolol On Proliferation Of Endothelial Cells Of Hemangioma

Objective:Infantile hemangiomas are the most common benign vascular tumors in infancy.They occur in 5-10%of the population,with females affected three times more often than males.Typically,infantile hemangiomas present shortly after birth,undergo a period of rapid proliferation and then slowly involute over many years.Although most are small cutaneous vascular malformations of the face,they can also be large,disfiguring lesions with serious complications.Infants with large hemangiomas,especially those with a segmental distribution or hemangiomatosis,are at particular risk for extracutaneous complications.Patients require active systemic therapies in critical conditions that may lead to complications such as disfigurement and scarring or,in more severe cases,visual impairment,airway compromise,congestive heart failure,and even death.The pathogenesis of infantile hemangiomas is not clear,in recent years,researchers make a large number of studies about pathogenesis of hemangioma from the cells,cytokines,genes,proteases and its inhibitors.The following theories are formed:（1）The imbalance of pro-angiogenic and inhibitory factors.VEGF（vascular endothelial cell growth factor）and b FGF（basic fibroblast growth factor）are two major pro-angiogenic factors.They are significantly increased in the proliferation of hemangiomas.（2）Apoptosis theory,apoptosis-related proteins participate in the process of hemangioma degradation by participating in endothelial cell apoptosis.（3）Activation of signal transduction pathway:（1）P13K/AKT;（2）Ras/MAPK;（3）NOS;（4）PKC;（5）FAK/Paxillin.The above signaling pathway has led to the process of vascular endothelial cell proliferation,cell survival,cell migration,cytoskeleton rearrangement,cell infiltration and other processes.Previously,the first-line treatment of infantile hemangiomas include laser treatment,surgery and medication.The only suitable treatment for early superficial hemangiomas is laser treatment.Due to facial nerve injury and scar formation and other complications,surgery must be carefully selected.Commonly used drugs used to treat hemangiomas are corticosteroids and anti-cancer drugs,but these drugs can cause varying degrees of side effects.In recent years,β-blockers have achieved good results in the treatment of hemangiomas,which has replaced corticosteroids as the first-line drug for the treatment of infantile hemangiomas and has drawn the attention of scholars from all over the world for a series of related studies on the treatment of infantile hemangiomas with beta-blockers.Since October 2012,more than 300 children with proliferative hemangioma in oral and maxillofacial surgery（Stomatological Hospital of China Medical University,Shenyang,Liaoning Province）have taken propranolol orally at a dose of 1.0-1.5 mg/kg/day.A small amount of 0.5%Timolol Maleate Eye Drops was applied to the lesion area with medical cotton swabs twice a day,once every 12 hours.Age ranged from 2 to 9 months,with an average age of 4.7 months.The lesions were located in the parotid gland,around the orbit,shoulder,ear and temple,and the volume ranged from 3.5x4x0.5 to 7x8x3cm.The treatment plan lasts for 6 to 8 months,or two drugs are discontinued after the lesion has completely disappeared.The results and safety of the treatment were assessed by changes in the size and color of the tumors and adverse reactions during the whole course of treatment.The average duration of treatment was 21.1 weeks,ranging from 3 months to 8months.Among them,72%showed good response and 0.09%showed moderate response.No major side effects were observed.In conclusion,oral propranolol combined with topical timolol maleate may be the first choice for the treatment of mixed hemangioma of parotid gland in infants and young children.This study attempts to use molecular biology methods,using different doses ofβ-receptor blocker timolol maleate in cultured human umbilical vein endothelial cells（HUVEC）,to explore its effect on cell proliferation and apoptosis and the mechanism of timolol in the treatment of hemangiomas.Meanwhile,to study the effect of timolol maleate combined with propranolol hydrochloride,anotherβ-receptor blocker was used in human umbilical vein endothelial cells（HUVEC）to investigate cell proliferation,apoptosis and other effects,providing a theoretical basis for clinical use of the combination of the two drugs.Methods:1.Cell Culture:HUVEC cells were cultured in DMEM high glucose medium containing10%fetal bovine serum,100 U/ml penicillin and 100 U/ml streptomycin,and cultured in a thermostatic incubator at 37℃and 5%CO₂concentration.2.CCK-8 detection of cell proliferation:5000 cells were seeded in 96-well plates for treatment with drugs,the corresponding wells were added 10μL CCK-8,and placed at37℃for 4 hours.Read the absorbance of each well at OD 450 nm using a microplate reader.3.Annexin V-FITC/PI double staining detection of cell apoptosis:3×10⁵cells were seeded in a 6-well plate.After drug treatment,the cells were harvested by trypsin without EDTA.Add 500μL of Binding Buffer to each sample for cells suspension.Add 5μL Annexin V-FITC and 5μL Propidium Iodide and mix well.Dark reaction at room temperature for 10min.The cells were removed by filtration through a 300 mesh sieve,and the apoptosis was detected by flow cytometry.4.Hoechst33258 detection of apoptosis:3×10⁵cells were seeded in a 6-well plate for drug treatment,aspirate the culture medium,add 1m L Hoechst33258 staining solution,placed in 37℃reaction for 30min.Discard staining solution,rinse with PBS and observe with fluorescence microscope.5.Tube formation assay:3×10⁵cells were seeded in a 6-well plate for drug treatment,then the cells were harvested.Take melted Matrigel 50μL spread in the wells of the96-well plate to avoid bubbles,placed at 37℃for 45min.After the matrigel has been solidified,the drug-pretreated cells that have been collected were seeded into a 96-well culture plate while continuing 10%fetal bovine serum and different concentrations of timolol maleate.After 6h,the cells were observed for tube formation using an inverted microscope.6.RT-PCR detection:1×10⁶cells were seeded in 60mm cultured dishes for drug treatment,RNA extraction kit was used to extract the total RNA of HUVEC cells.TAKARA’s Prime Script ^TMRT reagent reverse transcription Kit with g DNA Eraser were used to subject reverse transcription reaction to obtain the first strand c DNA.Finally,use SYBR Premix Ex Taq^TMfluorescence quantitative PCR kit to perform quantitative PCR reaction in quantitative PCR machine.According to the instructions of the PCR kit,the PCR reaction conditions are 95℃for 30s,95℃for 5s and 63℃for 20s,40 cycles.After the experiment,the results were analyzed by 2^-△△CTmethod.7.Western blot detection:1×10⁶cells were seeded in 60mm cultured dishes for drug treatment,RIPA lysate was used to extract the total cellular protein and the protein concentration was determined by BCA protein assay kit.ECL exposure was used to detect the expression and their respective phosphorylation levels of ERK,Akt,m TOR and FAK by polyacrylamide gel electrophoresis,transfer and antibody incubation.The expression of the growth factors VEGF and b FGF were also examined.8.Statistical Analysis:The experimental data were analyzed by using SPSS17.0statistical software,the results histogram were expressed by mean±standard deviation.One-way analysis of variance was used to compare the significance of the differences between the two groups.P<0.05 was statistically significant.Results:1.CCK-8 results showed when timolol maleate was applied to HUVEC for 24 h,the cell proliferation was significantly inhibited at 100μM,and the inhibitory effect was more significant with increasing concentrations of timolol maleate.After 48h treatment,the concentration of 50μM began to inhibit cell proliferation in a dose-dependent manner.2.Annexin V-FITC/PI double staining results showed that treated HUVEC cells with timolol maleate for 24h significantly promote apoptosis,early apoptosis and late apoptosis rate of cells increased with the concentration of the drug increasing.3.Tube formation experiments showed that HUVEC cells pretreated with timolol maleate for 24h were tested for tube formation,and the ability of tube formation after 6h was significantly weakened in a dose-dependent manner.4.The results of RT-PCR and Western blot showed that timolol maleate significantly inhibited the expression of ERK,Akt,m TOR and FAK in HUVECs induced by VEGF at24h,and inhibited the phosphorylation of various pathway proteins.5.CCK-8 results showed that the combination of timolol maleate and propranolol hydrochloride inhibited the proliferation of vascular endothelial cells significantly better than timolol maleate alone,with the concentration of the two combination drugs gradually increased,the inhibitory effect is more obvious,and the effect for treating 72h is better than 48h.6.Hoechst33258 staining results showed that the combination of timolol maleate and propranolol hydrochloride for 48h induced apoptosis in a dose-dependent manner.7.The results of RT-PCR and Western blot showed that the combination of timolol maleate and propranolol hydrochloride for 48h and 72h significantly inhibited the expression of VEGF and b FGF at the gene and protein levels in time and dose-dependent manner.Conclusion:1.Timolol maleate inhibits vascular endothelial cell proliferation and the formation of blood vessels and promote apoptosis to achieve the purpose of treating infantile hemangiomas.2.Timolol maleate inhibits the proliferation of vascular endothelial cells and induces apoptosis by inhibiting the gene expression of ERK,Akt,m TOR and FAK and the phosphorylation of these proteins.3.Timolol maleate combined with propranolol hydrochloride inhibits the expression of VEGF and b FGF,thereby inhibiting the proliferation of vascular endothelial cells and promoting apoptosis.