Study Of The Effects Of Propranolol And Its Enantiomers On Pericytes Of Infantile Hemangioma

BackgroundInfantile hemangiomas (IH) is a common benign tumor in infants, whose incidence rate is1%-2%and this rate is increasing year by year. IH often occurs in the head and neck, and the location is relatively superficial. Sometimes IH occurs in the mucosa, muscle, bone tissue and internal organs.It affects the appearance and the physiological function. Although IH is common, but its pathogenesis is unclear and the efficacy is imprecise. The serious effect of infantile hemangiomas is incurable to a surgeon. In2008, a French doctor first discovered propranolol (PPL) had a therapeutic effect to IH. From then on, PPL was paid close attention, and it has become the fisrt-line therapy drug to IH. People continue to explore the therapeutic mechanism of PPL, but the related researches were mainly in the research on the vascular endothelial cells of IH, the role of the pericytes of IH was still not concerned.Pericytes are important cellular components of infantile hemangiomas. Pericytes and endothelial cells together constitute a barrier between vas capillary and tissue. Pericytes can maintain capillary wall tension, inhibit the proliferation of endothelial cells, regulate microvascular blood flow and the growth of new blood vessels, maintain capillary permeability and capillary stability. Pericytes is an important factor to maintain a stable internal environment. We hypothesized that pericytes may play an important role in the disease process of IH.In addition, PPL is a classical chiral drugs, which has two enantiomers, the left-hand body S-PPL and the right-hand body R-PPL. Clinically it is administered in the form of the racemate. The racemate is a mixture of the left-hand body and the right-hand body. Pharmacological studies have shown that the enantiomers have different pharmacological pharmacolinetic properties, and the enantiomers should be treated as two drugs. The roles that the enantiomers of PPL played in the treatment mechanism of IH and the effects which enantiomers of PPL acted on pericytes have not been reported.ObjectiveFirst, to isolate pericytes from hemangioma specimens, then start to study how propranolol affects pericytes, to determine whether the hemangioma pericytes play a role in the process of propranolol therapy; to study how enantiomers of propranolol affects the protein synthesis in pericytes, and then provide a theoretical basis for further study about the role which the enantiomers of propranolol played in the treatment mechanisms of infantile hemangioma.MethodsThe first part:To obtain pericytes from hemangioma specimens, then purify, culture and subculture pericytes. The morphology of pericytes was observed and the growth curves of pericytes were drawed. The markers of pericytes, NG2, PDGFR-β, D146, a-SMA, CD31, CD34, CD45and SM22-a, were identified by flow cytometry. Use SPSS17.0statistical software to analyze all data, and the results were described as x±s.The second part:The effects of propranolol on pericytes proliferation were detected by CCK-8assay. After pericytes were cultured in the medium containing propranolol, F-actin protein in cells was detected by immunofluorescence staining. The contraction of pericytes induced by propranolol was detected by gel contraction experiment. After pericytes were cultured in the medium containing propranolol, the level of VEGF/bFGF in supernatants was detected by ELISA, and the level of mRNA of VEGF/bFGF in pericytes was detected by RT-PCR. Use SPSS17.0statistical software to analyze all data, and the results were described as x±s and analyzed by variance and t test. The third part:After pericytes were cultured in the medium containing different enantiomers of propranolol, the change of cellular protein synthesis was detected by IEF-SDS-PAGE two-dimensional gel electrophoresis. Then the patterns of two-dimensional gel electrophoresis were obtained. The images were analyzed by PDQuest7.4software, and the proteins which were expressed, differentially were obtained by analyzing MALDI-TOF peptide mass fingerprintings.ResultsThe first part:1、HE staining of infantile hemangioma:Vascular endothelial cells were dense in tissue slices of IH, the nucleus of vascular endothelial cells are large and light, partly growed like streak, built up the immature vascular lumens which were small and less.2、Cell morphology and growth pattern:The isolated cells were elongated diamond or polygonal, and had adherent growth, showing non-contact inhibition and overlap like growth characteristics. Cells were cultured for5to7days and then colonies were formed; cells were cultured for10days and a wide range of colony were formed. Primary cells growed slowly, needed14to21days to reach conjunction. But passaged cells needed6to7days to reach conjunction. The growth curve of passaged cell was measured by MTT method. The results showed the5-7d was logarithmic growth phase and the10-12d was plateau phase.3、The results of flow cytometry:Passaged cells may express the markers of primary cell stably, i.e. NG2(+)、PDGFR-β(+)、D146(+)、α-SMA(+)、CD31(-)、 CD34(-)、CD45(-)and SM22-α(-)。The second part:I The effects of propranolol on pericytes proliferation were detected by CCK-8assay.The pericytes proliferation was detected by CCK-8assay under different concentrations of propranolol. Six groups were set according to the concentration of propranolol,0、10、20、40、80、160μmol/L.0μmol/L propranolol was the control group, and others were the experimental groups. Based on the results, we got the inhibition rate curve. Inhibition rate curve showed the growth inhibition of pericytes was seen at the40umol/L propranolol group, and the strength of inhibition increased with the increasing of the concentration of pericytes. At the40μmol/L propranolol group, the morphology of pericytes was elongated diamond or polygonal, and cells were intact without rupture.40μmol/L was an ideal concentration of propranolol in this experiment.Ⅱ Influence of propranolol on pericyte contractions.1、The changes of F-actin protein in pericytes were detected by immunofluorescence staining.Pericytes were treated by40μmol/L propranolol in the experimental group, and pericytes were not treated by propranolol in the control group. When viewed under a fluorescent microscope, the fluorescence intensity of the experimental group was significantly better than the control group. The relative fluorescence intensity of F-actin (fold increase) were1.00±0.08(in the control group) and1.68±0.11(in the experimental group). The experimental group was about1.68times of the control group, and the difference was statistically significant (P<0.05).2、The gel contraction experiments of pericytes.The results of gel contraction experiments of pericytes showed that pericytes in both groups occurred contraction, but the contraction in the experimental group was more significant. The percent of gel area in the control group (85.21±4.36)%, while the percentage of gel area in the experimental group was (59.17±6.28)%, the difference was statistically significant (P<0.05).Ⅲ PPL affects the protein synthesis of VEGF/bFGF in pericytes.1、The changes of VEGF/bFGF in pericytes supernatant.The levels of VEGF/bFGF proteins in pericytes supernatant were detected by ELISA in the experimental group (40μmol/L propranolol) and the control group (no propranolol). The results showed that2207±57pg/ml, the protein content of VEGF in the supernatant, in the control group, was significantly higher than1682±148pg/ ml in the experimental group, and the difference was statistically significant (P <0.05); the protein content of bFGF in the supernatant in the control group was significantly higher than in the experimental group, and the difference was statistically significant (116.78±8.42pg/ml vs78.54±4.71pg/ml, P<0.05).2、The changes of VEGF/bFGF mRNA in pericytes.The levels of VEGF/bFGF mRNA in pericytes were detected by RT-PCR in the experimental group (40μmol/L propranolol) and the control group (no propranolol). The results were displayed by percentage, and the level of mRNA in the control group was set as100%. The results showed that the levels of VEGF mRNA in the experimental group [(69.71±8.49)%] was significantly lower than the control group [(100.00±1.31)%], the difference was significant (P<0.05); The levels of bFGF mRNA in the experimental group were significantly lower than the control group, the difference was significant [(63.81±4.22)%vs (100.00±2.76)%, P<0.05].The third part:1、The results of two-dimensional gel electrophoresis.Compare the two-dimensional gel electrophoresis patterns of R-Pro with S-Pro. The results shown823±16protein spots were detected, and the matching rate was78%. There were32proteins that were expressed differentially by the analysis of PDQuest7.4software,2、The result of mass spectrometry.32proteins which were expressed differentially in two-dimensional gel electrophoresis profiles were analyzed by mass spectrometry.21peptide fingerprintings were got and searched in SwissProt database by using Mascot software.11proteins were found and they were NFKB3、TGM2、NACGT1、 HSP90A,、HSP90B、BCL2、BKLHD1、MMP9、CYK18、RAB36and NFKBIA.Conclusion1、Pericytes were successfully isolated from infantile hemangioma specimens, and pericytes could be subculture stably.2、One therapeutic mechanism of propranolol to infantile hemangioma may be achieved by pericytes:Propranolol can inhibit the growth of IH by inhibiting proliferation of pericytes and increase F-actin protein synthesis and enhance the contractility of pericytes, then the blood flow is reduced by the capillary hemangioma shrink, at last, the volume of hemangioma is reduced; propranolol can inhibit the synthesis and paracrine of VEGF/bFGF of pericytes, then the growth of endothelial is inhibited because of reducing the stimulation of VEGF/bFGF. All those led to the death of endothelial cell and the inhibition of angiogenesis.3、R-PPL and S-PPL cause the different protein synthesis of pericytes isolated from IH. It is necessary to sudy each enantiomer of propranolol as a single drug in order to reveal the therapeutic mechanism of IH.