Study On The Function Of Lymphotoxins In Pathological Lymphangiogenesis And Angiogenesis

Part ?.The expression and the distribution of proteins of lymphotoxin signaling pathway in lymphatic malformationObjective: Lymphotoxin signaling pathway plays essential roles in autoimmune disease,inflammation and progression of tumors.Here,we aim to explore the expression and the distribution of lymphotoxins,lymphotoxin receptors and their down-stream molecules,and to further analyze the correlation between proliferation and activation of NF-?B pathways,in order to evaluate the role of NF-?B pathways in progression of lymphatic malformation(LM).Methods: The expression and the distribution of lymphotoxins,including LT-?,LT-? and LIGHT,and their receptors were detected by ELISA,immunohistochemistry and real-time PCR.The nuclear translocation of NF-?B transcriptional factors and the up-regulation of Ki67 were determined as well.At last,cluster analysis was performed to evaluate the correlation between proliferation of lymphatic endothelial cells and activation of NF-?B pathways in 26 clinical LM tissues.Results: Lymphotoxins were mainly expressed in tertiary lymphoid organs(TLOs)located in stroma of LMs.Compared with normal skin tissues,the increased expression of TNFR1,TNFR2 and LT?R was observed in LMs,especially in those with infection.Besides,the activation of NF-?B pathways was elevated in LMs.Results from cluster analysis revealed positive correlation among lymphotoxins,NF-?B transcriptional factors and Ki67.Conclusions: The expression lymphotoxins and their receptors was confirmed in LMs.The expression of downstream molecules in lymphotoxin pathways,including classical and alternative NF-?B pathway,and Ki67 was also up-regulated in LMs.The significantly positive correlation among proteins further indicated that,via NF-?B pathways,lymphotoxins could enhance the proliferation of lymphatic endothelial cells and further promote progression of LMs.Part ?.The establishment of rat lymphatic malformation model and the underlying mechanism in progression of lymphatic malformation promoted by lymphotoxin signalingObjective: In the present part,human dermal lymphatic endothelial cells(Hd LECs)was stimulated with lymphotoxins in vitro,in order to prove the function of lymphotoxin pathways in proliferation of Hd LECs directly.Meanwhile,establishing rat lymphatic malformation(LM)with subcutaneous injection of Freud's Incomplete Adjuvant in neck or mouth floor region is reproductive.With the treatment of Lipopolysaccharide(LPS),the model mimicked inflammation in LMs,providing powerful models to studying the underlying mechanism of lymphotoxin signal in progression of LMs.Methods: The activation of NF-?B pathways and proliferation was tested by immunofluorescence and western-blot.With the application of small interfering RNA(si RNA),the roles of lymphotoxin receptors in the pathways were determined.The rat LM models were established by subcutaneous injection of Freund's Incomplete Adjuvant in neck region.The correlation between lymphotoxin pathways and proliferation in lymphatic endothelial cells was further determined by immunohistochemistry.Results: The results from in vitro experiments suggested that lymphotoxins could activate NF-?B pathways in Hd LECs.LPS up-regulated the expression of lymphotoxin receptors in Hd LECs,and further enhanced responses of Hd LECs to lymphotoxins.Moreover,LPS increased the lesions of rat LM models.The lesions were further examined by immunohistochemistry.The results from immunohistochemistry revealed that LPS could increase the expression lymphotoxins,activated NF-?B signaling and stimulated proliferation of lymphatic endothelial cells in vivo.The application of neutralization antibodies targeting lymphotoxin signaling could inhibit the progression of LMs promoted by LPS.Conclusions: Both LPS and lymphotoxins could promote the proliferation in lymphatic malformation mainly in a TNFR1/LT?R-dependent manner.Further,inflammation-related lymphotoxins could enhance proliferation in lymphatic endothelial cells via NF-?B signaling and promoted the progression of LMs.Part ?.The source of lymphotoxin-? in head and neck squamous carcinoma and its functions,as well as underlying mechanisms,in tumor angiogenesisObjective: In the present study,tissue array and co-culture system were utilized to figure out the source of lymphotoxin-?(LT-?)in head and neck squamous carcinomas(HNSCCs).The functions of LT-? to glycolysis,proliferation,migration and tube formation was explored in endothelial cells.The underlying mechanisms were further determined.Methods: Tumor Immune Estimation Resource(TIMER)and HNSCCs tissue array were used to study the correlation between the expression of LT-? and the infiltration of immunocytes in HNSCCs.The co-culture of human HNSCC cells Cal27 and primary lymphocytes,including T cells and B cells,was established to explore the functions of tumor cells on infiltrated lymphocytes.Further,the roles of LT-? in tumor angiogenesis were examined by metabolism assays,Ed U incorporation,Transwell migration assay,cell cycle assay and tube formation assay.Wester-blot was used to figure out the underlying mechanisms.Results: The data from TIMER,together with tissue array,indicated the expression of LT-? positively correlated with infiltration of lymphocytes.According to the results form co-culture,Cal27 stimulated the secretion of LT-? in B cells and T cells.Moreover,LT-? could upregulated glycolysis in HUVECS,supported by increased glucose uptake,lactate production and ATP generation.The results from western-blots indicated the up-regulation of PFKFB3,a key enzyme in glycolysis,was attributed to glycolysis promoted by LT-?.Furthermore,LT-? could enhance the proliferation,migration,tube formation,cell cycle and podia formation in a PFKFB3-dependent manner.With the treatment of TNFR si RNA and NF-?B signaling inhibitor,NF-?B classical pathway was confirmed involved in regulation of PFKFB3.Conclusions: The lymphocytes infiltrated in HNSCCs could secrete LT-? followed by promoting tumor angiogenesis through up-regulating PFKFB3-dependent glycolysis.With upregulation of PFKFB3-dependent glycolysis,LT-? promoted proliferation by driving the cell cycle and enhanced migration by increasing podia formation in HUVECs.Part ?.Functional inhibition of PFKFB3 in endothelial cells impaired angiogenesis in tumor xenograftsObjective: With the application PFKFB3 competitive inhibitor PFK15,we studied the impact of PFKFB3 inhibition in angiogenesis,aiming to explore the potential of targeting PFKFB3 in inhibiting tumor angiogenesis.Methods: PFK15 was utilized to inhibition the function of PFKFB3.Based on this,its functions were tested in angiogenesis of HUVECs by metabolism assays,Ed U incorporation,Transwell migration assay,cell cycle assay and tube formation assay.Nude mice tumor xenograft was established to study the functions of PFK15 in vivo.The formation of microvessels in HNSCCs was studied by immunohistochemistry staining of CD31 in serial sections of fixed tumor xenografts.Results: PFK15 impaired the up-regulated glucose uptake,lactate production and ATP generation simulated by LT-?.The proliferation,migration and tube formation in HUVECs were also attenuated when PFK15 inhibited the function of PFKFB3.Further,PFK15 increased tumor size and weight in Cal27 xenografts.And the results from immunohistochemistry suggested that PFK15 dramatically decreased the formation of CD31 positive microvessles in vivo.Conclusions: Inhibition of PFKFB3 by PFK15 impaired angiogenesis in vitro and in vivo,indicating promising therapeutic value of PFKFB3-targeting angiogenesis inhibition in the treatment of HNSCCs.