The Clinical Value Of Vascular Endothelial Growth Factor D In Lymphangiomyomatosis And Preliminary Exploration Of Other Biomarkers

Part ? Analysis of Vascular Endothelial Growth Factor D Level and Clinical Characteristics of Lymphangioleiomyomatosis and Long-term Observation of Rapamycin EfficacyObjective:To analyze the correlation between serum vascular endothelial growth factor D(VEGF-D)level and clinical characteristics in patients with lymphangioleiomyomatosis(LAM),and to evaluate the long-term efficacy of rapamycin on level of serum VEGF-D and other clinical features.Methods:A retrospective analysis of the LAM patients with probable or definite diagnosis who registered in Peking Union Medical College Hospital from October 2005 to November 2017 was conducted.Baseline and long-term follow-up clinical data about serum VEGF-D level,lung function,blood gas analysis,six minute walk distance,Borg dyspnea index,St George's respiratory questionnaire(SGRQ),CT grading were collected.Results:Three hundred and ninety LAM patients were enrolled(368 of sporadic-LAM,22 of tuberous sclerosiscomplex-LAM).The mean first visit age was 39.319.6 yrs.The mean follow-up time was 2.7±2.3 yrs.The baseline level of VEGF-D was 2754.01±2621.16 pg/ml.Serum VEGF-D level was weakly correlated with chylothorax,the diffusing capacity of the lungs for carbon monoxide(DLCO)%predicted(%pred),forced expiratory volume in 1 second(FEV1)%pred,and CT grading(r=0.392,-0.206,-0.177,-0.177,P<0.05).One hundred and fifty two in 390 LAM patients were not treated with rapamycin,and the intraindividual variability of serum VEGF-D level ranged from-35.23%to 47.34%;the other 238 LAM patients were received rapamycin therapy with an average treatment time of 2.0±1.6 yrs.The serum VEGF-D level after treatment were significantly decreased compared with the baseline level(2083.66±2126.44 pg/ml vs.3719.55±3298.64 pg/ml,P<0.01),the average decline rate was 886.77±189.40 pg/ml/yr and remained low degree for a long time.Similarly,SGRQ and 6MWD were significantly improved after rapamycin treatment(P<0.01).In addition,the FEV1 decreased by 179.79±75.22ml/yr and DLCO decreased by 290.42±156.79 mmol/min/kPa/yr before rapamycin treatment,but during treatment FEV1 increased by 28.23±53.94ml/yr,and DLCO increased by 16.90±79.12mmol/min/kPa/yr.The long-term trend showed that both FEV1%pred and DLCO%pred remained stable within 3 years after treatment,but showed a significant decline after 3 years.The change of VEGF-D level was not parallel to FEV1%pred and DLco%pred.Conclusions:1.Serum VEGF-D in LAM patients is faintly correlated to clinical features,lung function and CT grading.2.Rapamycin significantly reduces serum VEGF-D levels,improves or stabilizes lung function,and accompanies with improvement in clinical symptoms in LAM patients.3.The change of serum VEGF-D level is not parallel to the change of lung function.Rapamycin can continuously reduce the serum VEGF-D level,but its long-term stability on lung function was not achieved from our data.Part ? Exploration of plasma potential biomarkers in patients with lymphangioleiomyomatosis based on MicroRNA sequencingObjective:To investigate the differential expression of plasma miRNA in patients with lymphangioleiomyomatosis(LAM)by microRNA sequencing,and to analyze its biological function.Methods:Seventheen sporadic LAM patients who participated in the LAM registry from April 2017 to November 2017 were collectedin the experimental group.We excluded the patients if they were treated on rapamycin,or they have pneumothorax,chylothorax,renal angiomyolipoma and other complications.Based on forced expiratory volume in 1 second(FEV,)%predicted(%pred),the experimental group was divided into FEVi%pred ? 70%sub-group with 10 patients and 50%?FEV1%pred<70%sub-group with 7 patients.Ten female healthy volunteers were enrolled in the control group.Plasma samples were taken for microRNA sequencing.Results:No statistical difference in mean age among different groups(44.00 ±11.08yrs vs.42.50 ± 8.83yrs,P<0.05).MicroRNA sequencing analysis showed that compared with the control group,there were 53 miRNAs upregulated and 217 miRNAs downregulated in the experimental group.ANOVA analysis showed that compared with the control group,miR-324-3p was the most significant difference miRNA(P<0.001),and it was downregulated by 1.54 fold change in the FEVi%pred?70%group and much higher with 3.41 fold change at 50%?FEV1%pred<70%group.The gene ontology(GO)enrichment analysis showed the differential miRNA target genes were mostly enriched in the "regulation of transcription" biological process,the "membrane" cellular component,and the "protein binding" molecular function,with a percentage of 77.35%,79.36%,90.90%,respectively;KEGG pathway enrichment analysis showed that there were 514 target genes enriched in"pathways in cancer",which ranged first,and 148 genes enriched in mTOR pathway(P<0.01).The KEGG pathway analysis of miR-324-3p target genes also showed most genes enriched in "pathways in cancer"(P?0.01).No target gene was found in the mTOR pathway.Protein-protein interactions showed the nodes of UBC,GAPDH,and HSPA8 in miR-324-3p target genes was the highest,being 54,50,and 49,respectively,which were most closely related in the protein interaction network map.Conclusions:1.There are a large number of differentially expressed miRNAs in plasma of LAM patients,and biological functions are mainly focused on cancer pathways,such as intracellular signal transduction,protein binding.2.The most significant differential expression on MicroRNA sequencing in LAM is miR-324-3p and it mainly involved in intracellular metabolic processes.There was no clear correlation between miR-324-3p and mTOR signaling pathway.