| Implications and objectiveHepatopulmonary syndrome normally occurs in patients who were sufferred with chronic liver disease in late stage,the main clinical symptom is hypoxemia.Based on the clinical statistics,the morbidity of HPS patients now are up to 4%-29%.At present,the research about HPS has been improved,howover,the mechanism of HPS is unclear.In addition,the transformation of clinical research should be in process.Actually,abnormal vascular dilation,arteriovenous shunt and the dysfunction of oxygenation are the mainly pathology of HPS,which are followed by the hypoxic pulmonary vascular remodeling,and then finally accelate the pathology in lung.In fact,the vascular dilation in pulmonary has been the central issue.In our previous study,the proliferation of pulmonary microvascular endothelial cells(PMVECs)may be related to vascular dilation.In addition,we find that the vascular resistance in lung is significantly increased in HPS patients after the treatment of liver tansplanation,which indicates the contraction of pulmonary microvasclar.Generally speaking,the pulmonary microvascular can not contract.Taking together,the proliferation of PMVECs can not result in the vascular dilation directly,some other pathological mechanism may take responsibility for it.In previous study,we found that the expression of SM-?-actin?calponin?SM-MHC were significantly increased in PMVECs when exposed to the HPS serum,which indicated the myogenic differentiation and potential contraction of PMVECs.Importantly,some researches have reported that Bone morphogenic protein-2(BMP-2)played a key role in the proliferation,differentiation,migration in many type of cell.But the role of BMP-2 in myogenic differentiation of PMVECs is unclear.Thus,this study plans to demonstrate that HPS serum induced BMP-2/Smad signaling pathway promotes the myogenic differentiation of PMVECs.Methods1.Rat model and serum preparation,Establishment of primary culture PMVECs,inducing myogenic differentiation of PMVECs under the condition of HPS serum.The HPS rat model was performed by chronic bile duct ligation(CBDL)in this study.After 28 days later,the efficacious HPS rats were identified by blood gas analysis and pathological section,and then HPS rats serum was gathered.The primary PMVECs culture were induced by tissue method,which were identified by immunohistochemistry.Under the condition of 5% HPS serum,PMVECs were clutured for 0h?24h?48h?72h,respectively,the morphology change were observed.And the expression of SM-a-actin,caplonin and SM-MHC were detected by western bloting.2.The effect of HPS serum in BMP-2/Smad signal pathway in PMVECs.The PMVECs were divided into two groups: control group and HPS group.PMVECs in both group were cultured under the condition of 5% control serum or 5% HPS serum for 24h?48h?72h,respectively.And the mRNA level and protein level of BMP-2 were detected by RT-PCR and western blot in various time point in each group.The expression level and activation level of Smad1/5,which was the downstream of BMP-2,were detected by western blot at various time point in each group.3.The effect of recombinant Noggin in the HPS induced myogenic differentiation of PMVECsRcombinant Noggin(50 ng/ml)was been used to inhibit the BMP-2/Smad signal pathway in PMVECs under the condition of 5% control serum or 5% HPS serum for 72 h,and then the change of Smad1/5 and P-Smad1/5 expression were detected by western blot.Meanwhile the expression of SM-a-actin,caplonin and SM-MHC were detected by western blot and imunohistochemistry.4.The effect of Smurf-1 mediated Smad1/5 degradation in HPS induced activation of BMP-2/SmadThe PMVECs were divided into two groups: control group and HPS group.PMVECs in both group were cultured under the condition of 5% control serum or 5% HPS serum for 24h?48h?72h,respectively.The expression of Smurf-1 was detected by western blot at various time point in each group.After that,Smurf-1 siRNA was trasnfected into PMVECs to downregulate the expression of Smurf-1.These Smurf-1 – silenced PMVECs was cultured under the condition of 5% control serum or 5% HPS serum for 72 h,and then the change of Smad1/5 and P-Smad1/5 expression were detected by western blot.Meanwhile,MG132 was used to inhibit the proteasome in PMVECsunder the condition of 5% control serum or 5% HPS serum for 72 h,and then the change of Smad1/5 and P-Smad1/5 expression were detected by western blot.Results1.HPS serum induces the myogenic differentiation of PMVECs.Before the stimulation of HPS serum,PMVECs were arranged like a spindle or polygonal.After being stimulated for 72 h,PMVECs were arranged fusiform.At the time point of 0 h,we found negative expression expression of SM-MHC,SM-?-actin,and calponin protein.After being stimulated for 72 h,the expression of SM-MHC,SM-?-actin,and calponin protein were significantly increased in a time depended manners compared with the time point of 0 h(P < 0.05).2.HPS serum activated the BMP-2/Smad signal pathway in PMVECs.In HPS group,both the expression levels of BMP-2 mRNA and protein in the PMVECs are obvious increased in a time depended manners compared with Control group(P < 0.05).Meawhilem,the expression of Smad1/5 and P-Smad1/5 are also upregulated compared with Control group(P < 0.05).3.The myogenic differentiation of PMVECs was inhibited partialy by NogginHPS serum induced the phosphorylation of Smad1/5,a process that was attenuated by the Noggin treatment(P<0.05).Notably,these events were followed by the down-regulation of both SM-?-actin and calponin,compared with the Noggin untreated Group HPS(P<0.05).Interestingly,Noggin had no effect on the expression of SM-MHC in Group HPS(P>0.05).Furthermore,immunostaining results also revealed this results.4.HPS serum inhibits Smurf-1 mediated degradation of Smad1/5,and promotes the activation of BMP-2/Smad signaling pathway.Compared with the control group,the expression of Smurf-1 in PMVECs exposure to HPS serum was significantly decrease at each time point(P<0.05);In the Smurf-1-silenced PMVECs,both Smad1/5 and P-Smad1/5 protein levels were higher than those obtained in the control si RNA-transfected cells.Furthermore,Smurf-1 knockdown enhanced both Smad1/5 and P-Smad1/5 protein levels in the presence of the CBDL-rat serum treatment(P<0.05).On the other hand,compared to both MG132 untreated Group HPS and the MG132 pretreated Group C,the expression levels of both Smad1/5 and P-Smad1/5 were significantly increased in MG132 pretreated Group HPS(P<0.05).ConclusionsOur findings suggest that BMP-2 regulates the myogenic differentiation of PMVECs induced by the CBDL-rat serum.Both the activation of P-Smad 1/5 and the down-regulation of Smurf-1 play the important roles in this process. |
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