Observation On The Efficacy Of Tumor Necrosis Factor-α Inhibitors In Patients With Livedoid Vasculopathy And Study Of Its Pathogenesis Based On Transcriptomic

BackgroundLivedoid vasculopathy(LV)is a relatively rare thrombo-occlusive cutaneous vascular disease,characterized by dermal microcirculation thrombosis.It is estimated that the incidence of LV is about 1:100000.At present,because of the unclear etiology and pathogenesis,the treatment of LV is still controversial and challenging,which seriously impaires on their quality of life.Rivaroxaban is the major treatment for LV patients.However,previous studies have found that monotherapy of anticoagulant may not achieve favorable clinical response,and anti-inflammatory medications are also indispensable for LV at acute ulcerative stage.In recent years,biological agents have been widely used in the treatment of skin diseases like psoriasis,autoimmune bullous disease,atopic dermatitis,hidradenitis suppurativa and pyoderma gangrenosum.There have been case reports of biological agents and small molecule targeted drugs such as tumor necrosis factor(TNF)a inhibitors being effective for refractory LV patients.Whereas,research on the efficacy and safety of TNF-α inhibitors in treating LV patients is limited.And the specific mechanism and therapeutic effect of TNF-α inhibitors in LV is unknown,which warrants further research.In addition to hypercoagulable thrombotic factors,the pathogenesis of LV may also involve immune,metabolic,and inflammatory factors.Previous studies on the pathogenesis of LV focused on multiple coagulation related single nucleotide polymorphisms,the abnormalities of plasminogen activator inhibitor-1(PAI-1),and microarray transcriptome research based on RNA gene chips.Transcriptome research reflects the sum of mRNA sequences transcribed in the functional state limited by specific time and space conditions.Previously,it has shown great scientific value and research potential in a variety of inflammatory skin diseases and skin tumors.Up to now,there has been no high-throughput transcriptome research for LV patients.Based on the results of transcriptome and PAI-1 which is known to play an important role in the pathogenesis of LV,this study will search for possible upstream factors in thrombosis and inflammation,and further study the role of high mobility group box 1(HMGB1)as a damage related molecular model and stress response related protein in the pathogenesis of LV.Objective1.To carry out real-world data on efficacy and safety of TNF-α inhibitors in refractory LV patients,and to conduct analysis on platelet related parameters in LV patients to explore clinical biomarker for predicting disease activity,recurrence and differential diagnosis.2.To explore the pathogenesis of LV based on high-throughput transcriptome sequencing technology.3.To detect the expression of HMGB1 and its related downstream factors in LV patients,and to investigate the effect of HMGB1 on human microvascular endothelial cell(HMEC)1 in vitro.Methods1.A total of 11 LV patients were included to receive recombinant human type II TNF-αantibody receptor fusion protein 50mg once a week for subcutaneously injection.The Numerical rating scale(NRS),LV composite clinical score,and Dermatology life quality index(DLQI)were used to evaluate the efficacy at the 2nd,4th,8th,and 12th weeks.2.Compare the platelet related parameters of acute active LV patients(n=27)and cutaneous small vessel vasculitis patients(n=21),acute active LV patients(n=20)and stable LV patients(n=20),and analyze the correlation between platelet related parameters and LV composite clinical score.3.High-throughput transcriptome sequencing was carried out in skin tissues of LV patients(6 samples).According to bioinformatics analysis,differential expression genes were screened,and verified by real-time quantitative polymerase chain reaction(RT-qPCR).4.Expression of HMGB 1 and its downstream factors PAI-1,vitronectin(VN)and hypoxia inducible factor(HIF)-1α were detected by RT-qPCR and ELISA in skin tissue(n=5)and plasma(n=21)of LV patients.5.Detect PAI-1 4G/5G gene polymorphism using genomic DNA from peripheral blood in LV patients(n=19),and analyze the association between genotype and clinical characteristics,coagulation function,and hypercoagulable thrombotic factors in LV.6.In vitro experiment,HMEC-1 cells were treated with recombinant human HMGB1 protein at concentrations of 20ng/ml,50ng/ml,and 100ng/ml,respectively.Cell proliferation and viability were detected using the CCK8,and the expression of advanced glycation end products(RAGE)and Toll like receptor 4(TLR4)was detected using RTqPCR method.The expression of PAI-1,TNF-α,HIF-1α and vascular endothelial growth factor(VEGF)in supernatant of cell culture medium were detected using ELISA method.Results1.TNF-α inhibitors showed favorable response on pain relief,ulcer healing,and improvement on quality of life in both idiopathic LV patients and patients with hypercoagulability and thrombotic factors,which provide early and rapid pain relief with good tolerance.Long term therapy contributes to the prevention of disease recurrence and exacerbation,especially in summer.2.The platelet count and plateletcrit(PCT)levels in LV patients were significantly higher than those in patients with cutaneous small vessel vasculitis.The platelet count and PCT levels in LV patients at acute active stage were significantly higher than those at stable stage.The composite clinical score of LV was positively correlated with platelet count and PCT levels.3.There were 78 differentially expressed genes between lesional skin tissues and perilesional skin tissues through high-throughput transcriptome sequencing technology,of which 51 genes were up-regulated and 27 genes were down-regulated.According to bioinformatics analysis,differential expression genes were screened,and the RT-qPCR method was used to detect the expression levels of FGA,RBP2,IGF2,IL17REL and SELE genes to verify the sequencing results.4.The expression of HMGB1 mRNA in skin tissues from lesional area in LV patients was significantly increased,and the plasma levels of PAI-1,VN,and HIF-1α were significantly increased.5.The PAI-1 4G/5G heterozygous genotype is the most common genotype in LV patients.LV patients in 4G group(4G/4G and 4G/5G genotype)showed a younger onset age,and the proportion of female patients is higher than that in 5G group(5G/5G genotype).The fibrinogen and D-dimer levels in LV patients in 4G group are higher than those in 5G group,while thrombin time and protein C levels are lower.6.The recombinant human HMGB1 protein at the above concentrations has a promoting effect on the proliferation and viability of HMEC-1 cells in the early 24 hours.RAGE mRNA levels were upregulated at the early 24 hours,while TLR4 mRNA expression showed an increasing trend at 48 hours.Expression levels of PAI-1,TNF-α,HIF-1α and VEGF in supernatant of cell culture medium treated with high concentration human recombinant HMGB1 protein(50ng/ml and 100ng/ml)were significantly increased in the early 24 hours.Conclusion1.TNF-α inhibitor is effective for acute active refractory LV patients with early and rapid pain relief.It is well-tolerated without severe adverse events during follow-up,providing a new treatment option for refractory LV patients.2.The altered platelet morphology is detected in LV patients.Platelet count and PCT may be clinical biomarker for predicting disease recurrence,activity and differential diagnosis.3.Through the analysis and comparison of the transcripts of skin tissues from LV patients,multiple metabolic related pathways and platelet activation pathways were enriched,and some differential expression genes between individual sample groups were enriched to inflammatory response related pathways,providing new insights into the pathogenesis and potential targets for treatment from the transcriptome perspective.4.The expression of HMGB1 and its downstream factors were increased in LV patients.HMGB1 revealed differential effects on cell proliferation,thrombosis and inflammatory responses via RAGE/TLR4 in vitro.HMGB1 plays an important role in the pathogenesis of LV at acute active stage,and provides theoretical basis for TNF-α inhibitor therapy and anti-inflammatory therapy of LV patients at acute active stage.