Podoplanin Mediate Islet Stellate Cells Activation Via Cell Deformation

PART Ⅰ.Podoplanin is overexpressed in activated ISCsObjective:We have demonstrated that activated islet stellate cells(ISCs)cause islet function impairment and induce islet fibrosis.Inhibition of ISCs activation is beneficial for preserving islet function,and is potentially a pivotal strategy in the prevention of diabetes.However,the key molecules responsible for ISCs activation have not yet been determined.Through RNA-sequencing and differentially expressed genes analysis,we found that the Pdpn gene was significantly overexpressed in ISCs of GK rats with severe islet fibrosis.This part intends to further verify the expression of Pdpn gene in a variety of activated ISCs models,and provide basic support to explore the role of Pdpn gene in the activation of ISCs.Methods:The ISCs from db/db,db/m,high fat diet(HFD)rats and normal diet(ND)diet rats were used in this part.The mRNA and protein expression levels of Pdpn or podoplanin(PDPN)in db/db-ISCs and db/m-ISCs,HFD-ISCs and ND-ISCs were detected by qRT-PCR and Western blotting,respectively.The expression levels of PDPN in pancreatic islets of db/db and db/m mice,HFD rats and ND rats were detected by immunofluorescence.All data were presented as mean ± standard error,Student’s T test was used for comparisons between two groups,and univariate ANOVA analysis was used for comparisons with three or more groups.P<0.05 was a significant difference.Results:1.In the ISCs of db/db mice,the mRNA(1.00±0.09 vs.2.27±0.21,P<0.01)and protein(1.00±0.03 vs.1.66±0.13,P<0.01)expression levels of Pdpn were significantly higher than those of db/m mouse ISCs.2.In ISCs of HFD rats,the mRNA(1.52±0.05 vs.1.00±0.05,P<0.01)and protein(1.86±0.12 vs.1.00±0.05,P<0.01)expression levels of Pdpn gene were significantly higher than those of ND rat ISCs.3.It was found by immunofluorescence that the expression of PDPN in the pancreatic islets of db/db mice and HFD rats were significantly higher than those of db/m mice and ND rats(db/db vs.db/m,1.38±0.44 vs.0.04±0.01,P<0.05;HFD vs.ND,1.15±0.29 vs.0.08±0.03,P<0.05).Conclusion:The PDPN expression in db/db mice and HFD rats ISCs were higher than those in normal controls.Combined with the early evidence in GK-ISCs,it was confirmed that the PDPN expression in ISCs was generally increased in Type 2 diabetes mellitus(T2DM)animals,suggesting that it may be related to the activation of ISCs in T2DM.PART Ⅱ.PDPN knockdown inhibited ISCs activation in miceObjective:Through RNA-sequencing and differentially expressed genes analysis,we found that the Pdpn gene was significantly overexpressed in ISCs of GK rats.Combined with validations in multiple T2DM animals,we hypothesized that the Pdpn may be the key gene responsible for the activation of ISCs.The role of Pdpn gene in the activation of ISCs needs to be further verified in vivo.Methods:In this part,an adeno-associated virus with a GFAP-specific promoter was constructed to knock down the Pdpn gene in stellate cells(GFAP-AAV-Pdpn-),as well as the control virus GFAP-AAV-con.7-week-old male C57BL/6J mice were selected and randomly divided into 6 groups(n=8 in each group).After one week of adaptive feeding(week 8),mice were selected for tail vein injection of GFAP-AAVPdpn-virus or GFAP-AAV-con virus(3*1011 v.g of virus injected into each mouse).After 3 weeks,the virus expression was stabilized and then low-dose STZ and highfat diet were used to establish T2DM models,namely CON,CON+AAV-con,CON+AAV-pdpn-,T2DM,T2DM+AAV-con and T2DM+AAV-pdpn-.Then the effect of virus was detected,and the body weight and fasting blood glucose of each group of mice were dynamically observed.Intraperitoneal glucose tolerance tests(IPGTT)and insulin tolerance tests(IPITT)were used to evaluate the glucose metabolism of mice in each group.The mice were sacrificed after 30 weeks,and the pancreatic sections were subjected to masson and immunofluorescence to evaluate the islet morphology,insulin,islet fibrosis area and the expression level of α-smooth muscle actin(α-SMA)in the islets of the mice in each group.The expressions of α-SMA and Col-Ⅲ(Collagen-Ⅲ,Col-Ⅲ)in the pancreas of mice in each group were investigated by Western blotting experiment.Results:1.It was found that the expression of PDPN in the pancreas of T2DM(1.49±0.12)and T2DM+AAV-con group(1.20±0.14)was significantly higher than that of control group(0.68±0.06,P<0.01),and the PDPN expression of T2DM+AAV-pdpn-group(0.33±0.06)was significantly lower than that of T2DM group(P<0.01).2.The body weight in CON,CON+AAV-con,CON+AAV-pdpngroups increased with the feeding time;The body weight of mice in T2DM,T2DM+AAV-con and T2DM+AAV-pdpn-groups increased slowly,and was significantly lower than the CON groups.There was no statistical difference within the T2DM,T2DM+AAV-con and T2DM+AAV-pdpn-groups.3.The fasting blood glucose of T2DM+AAV-pdpn-group(11.20±2.18)was significantly lower than that of T2DM group(17.53±1.88)and T2DM+AAV-con group(16.83±1.58,P<0.05).4.The IPGTT experiment found that the area under the AUC curve of T2DM+AAV-pdpn-group had a decreasing trend than T2DM group(4574±82.53 vs.5119±299.80,P=0.07).At 180 minutes,the blood glucose in the T2DM+AAVpdpn-group was significantly lower than that in the T2DM group(24.47±2.23 vs.17.43±0.81,P<0.05).5.There was no significant difference in the area under the IPITT AUC curve between T2DM+AAV-PDPN-group and T2DM group(2360 ±182.70 vs.2498±369.70,P>0.05).6.Immunofluorescence experiments showed that the mean fluorescence intensity of insulin in islets in T2DM+AAV-pdpn-group(142.10±4.24)was significantly higher than that in T2DM group(107.30±8.44)and T2DM+AAV-con group(109.7±5.33,P<0.05).7.The results of masson staining showed that compared with T2DM(2.96±0.38)and T2DM+AAV-con groups(3.07±0.40),T2DM+AAV-pdpn-group(1.877±2.01)had significantly less fibrotic areas in islets(P<0.05).8.Through Western blotting and immunofluorescence experiments,it was found that the expression of α-SMA and Col-Ⅲ in the pancreas and islets of the T2DM+AAV-pdpn-group was significantly lower than that of the T2DM and T2DM+AAV-con groups.Conclusion:Specific knockdown of the Pdpn gene in stellate cells inhibited the activation of ISCs in T2DM mice,suppressed the occurrence and development of islet fibrosis,and improved the function of islet β cells in mice.PART Ⅲ.The effect of PDPN on ISCs activation in vitroObjective:The expression of Pdpn is up-regulated in various activated ISCs cell models.Specific knockdown of Pdpn in stellate cells of T2DM mice can inhibit the activation of ISCs,inhibit the occurrence and development of islet fibrosis,and improve the function of islet β cells.The role of Pdpn in the activation of ISCs has not been elucidated in detail.This part will explore the role of Pdpn in the activation of ISCs in vitro.Methods:Small interfering RNA of Pdpn(siRNA-Pdpn)and control siRNANegative control(siRNA-NC),and Pdpn overexpression plasmid and control plasmid were constructed,and transfected into GK-ISCs(GK-ISCPdpn-,GK-ISCNC)and Wistar rat ISCs(W-ISCPdpn+,W-ISCNC).ISCs were simulated by advanced glycation endproducts(AGEs).The morphological changes of cells in each group were observed,and the expressions of PDPN,α-SMA,ECM and other proteins in cells of each group were detected by qRT-PCR,Western blotting,and cellular immunofluorescence.The changes in the proliferation and migration levels of cells in each group were explored by CCK-8,wound healing and Transwell experiments,and the rate of lipid droplet disappearance in each group was identified by Oil Red O staining.Results:1.The morphology of GK-ISCPdpn-changed from activated state to resting state cells,that is,from spindle cells with more pseudopodia to round cells with fewer pseudopodia.2.QRT-PCR,Western blotting,and cell immunofluorescence experiments showed that compared with GK-ISCNC,the expression levels of α-SMA,Col-Ⅰ,FN and other extracellular matrix components of GK-ISCPdpn-were significantly decreased,and the proliferation of cells was significantly reduced.With a marked decrease in the level of migration,the rate of lipid droplet disappearance also slowed down.3.The expression of PDPN,α-SMA and Col-Ⅰ was significantly increased in W-ISCs when AGEs interfered.4.Combined with AGEs and siRNAPdpn intervention,it can be found that interfering with Pdpn expression can reduce the mRNA and protein expression of AGEs-induced ISCs α-SMA and Col-Ⅰ.5.The cell morphology of W-ISCPdpn+changed from a relatively resting state to an activated state phenotype.6.QRT-PCR,Western blotting,and cell immunofluorescence experiments showed that compared with W-ISCNC,the gene transcription and protein expression levels of α-SMA and Col-Ⅰ,FN and other extracellular matrix components of W-ISCPdpn+were significantly increased.The level of proliferation and migration of the lipid droplets also increased significantly,and the disappearance rate of lipid droplets also increased significantly in W-ISCPdpn+cells.Conclusion:1.Down-regulation of Pdpn gene induced GK-ISCs to change from active state to resting state,decreased proliferation and migration ability,slowed down the disappearance rate of lipid droplets,and decreased the expression of α-SMA and ECM 2.Overexpression of Pdpn changes the ISCs from quiescent state to activated state,promotes the expression of α-SMA and ECM,and induces cell proliferation,migration and disappearance of lipid droplets.3.Knockdown the Pdpn expression can prevent and reverse the activation of ISCs induced by AGEs-simulated high-glucose environment.PART Ⅳ.PDPN/TGF-β1/SMAD2/3 autocrine ring promotes cell deformation and mediates ISCs activationObjective:Previously,it was shown that the expression of Pdpn is up-regulated in activated ISCs,and specific knockdown of Pdpn expression in vivo can inhibit islet fibrosis in T2DM mice.In vitro experiments confirmed that overexpression of Pdpn gene makes ISCs transition from a resting state to an activated state,promote the expression of its α-SMA and ECM.However,the mechanism has not been elucidated.This part of the study will elucidate the subcellular and molecular mechanisms of PDPN in the activation of ISCs.Methods:The ROCK kinase inhibitor Y-27632 was used to intervene W-ISCPdpn+and W-ISCNC,and the cell morphology changes were observed by microscopy and phalloidin staining,respectively.The expressions of PDPN,α-SMA,Col-Ⅲ,FN and other proteins in each group were detected by Western blotting and cell immunofluorescence,and the migration rate of ISCs in each group was detected by wound healing and Transwell assays;The expression levels of transforming growth factor-β1(TGF-β1)were detected in cells and tissues by Elisa.And ISCs were intervened by TGF-β1 or combined with TGF-β1 receptor Ⅰ(Transforming growth factor-β Receptor Ⅰ,TGF-βRI)inhibitor.Then western blotting was used to detect the expression of PDPN in cells of each group and the related signaling pathways of ISCs activation.Results:1.Compared with W-ISCPdpn+with more pseudopodia and longer pseudopodia extension,the pseudopodia of W-ISCPdpn+cells combined with ROCK kinase inhibitor disappeared,the shape tends to be round or triangular,and the cells became inactive state.ROCK kinase inhibitor can reduce the expression of α-SMA(0.69±0.07vs.1.48±0.11,P<0.01),FN(0.72±0.08vs.2.34±0.19,P<0.01)and reduce the cell migration ability of ISCs.2.The expression of TGF-β1 in the pancreas of T2DM mice was significantly higher than that of control.The expression of TGF-β1 in the pancreas of T2DM mice injected with GFAP-AAV-Pdpn-virus was significantly lower than that of T2DM and T2DM mice injected with AAV-con control virus.3.In ISCs cells cultured in vitro,we found that knockdown the PDPN could reduce the secretion and expression of TGF-β1,and overexpression of PDPN could increase the secretion and expression of TGF-β1.4.Adding TGF-β1 cytokine to the cell supernatant,it can be found that the expression levels of P-smad2/3 and PDPN in ISCs are up-regulated in a concentration-dependent manner.Adding different concentrations of TGF-βRI inhibitor,the expression levels of P-smad2/3 and PDPN were down-regulated in a concentration-dependent manner.5.PDPN can also enhance the activity of ISCs by increasing the phosphorylation level of ERK1/2.Conclusion:PDPN mediates ISCs activation via cell deformation and promotes TGFβ1 secretion,which in turn induces PDPN expression and ISCs activation by binding TGF-βRI to activate TGF-β1/SMAD and ERK/AKT signaling pathways.