Transmembrane Protein ESDN Regulates Smooth Muscle Cell Proliferation And Vascular Hyperplasia Through Modulate PDGF Signaling Pathway

Objective:Vascular hyperplasia is a common pathophysiological process in most of cardiovascular remodeling diseases,such as atherosclerosis?AS?,hypertension,restenosis after angioplasty.Vascular smooth muscle cells?VSMCs?,located in the media of aorta,maintain the contraction phenotype under the normal physiological state,which convert into synthetic phenotype after vascular injury.Platelet-derived growth factor?PDGF?-BB is released by platelets,endothelial cells?ECs?,VSMCs and inflammatory cells at the sites of vascular injury.Also,PDGF-BB is a particularly phenotypic switch growth factor for VSMCs and triggers VSMC proliferation and migration,which are the key cellular events in the process that ultimately leads to vascular remodeling and intimal hyperplasia.Endothelial and smooth muscle cell-derived neuropilin-like protein?ESDN?is a type I transmembrane protein with a CUB domain and coagulation factor V/VIII domain,which is similar to neuropilins.ESDN was initially cloned from human coronary arterial and highly metastatic lung cancer cells.Our previous study showed that ESDN can be induced by PDGF-BB and serum and is highly expressed in the neointima after injured.Furthermore,ESDN is up-regulated in proliferating VSMCs,and ESDN overexpression inhibits VSMC growth.ESDN knockdown can up-regulate PDGF-BB-induced DNA synthesis and cell proliferation.However,the mechanism by which ESDN regulates the PDGF receptor-?(PDGFR-??8?signaling pathway to affect vascular hyperplasia is still unclear.In this study,the smooth muscle cell conditional Esdn konockout mice that we generated were be used to the common carotid artery ligation animal model.Cultured VSMCs induced by PDGF-BB were be used to detect the effects of ESDN on PDGF signaling pathway and PDGFR-? endocytosis.Methods:1.Generation of the smooth muscle cell conditional Esdn knockout mice and genotypingSmooth muscle cell conditional Esdn knockout mice were generated using Esdn^flox/flox?kindly gifted from Dr.Mehran Sadeghi,Yale Medical School?and Tagln-cre?also called SM22aCre,The Jackson Laboratory?transgenic mice express Cre recombinase under the control of the mouse SM22apromoter.Mice of 6 to 8 weeks old were selected and mated at a ratio of 2:1?female:male?.The genomic DNA was extracted from the ears of mice and their genotypes were identified by polymerase chain reaction?PCR?.The Cre^+/-Esdn^flox/floxlox/flox and their control littermates Cre^-/-Esdn^flox/floxlox/flox mice were successfully generated.Protein extracts from aorta was also performed to verify the knockout effect by Western blotting.2.Preparation and grouping of mouse intimal hyperplasia modelThirty WT,and Esdn^-/-mice or Cre^+/-Esdn^flox/floxlox/flox and their control littermates Cre^-/-Esdn^flox/floxlox/flox mice were randomly divided into five different groups?control,3 days,7 days,14 days and 21 days after ligation group?.The intimal hyperplasia model was generated by ligation of the left common carotid artery under the microscope under the isoflurane inhalation anesthesia,the control?sham-operated?group only isolated blood vessels,without any ligation.The left common carotid arteries were harvested after perfusion with saline at 3,7,14,21 days after ligation,respectively,and then each one was divided into two different parts,one part embedded with OTC embedding agent,liquid nitrogen frozen quickly for immunofluorescence staining,another part was used the RIPA buffer?50 mM Tris-cl pH 7.5,150 mM NaCl,0.5%Deoxycholate,1%NP-40,0.1%SDS,1 mM EDTA,complete proteinase inhibitor from Roche Applied Sciences and phosphatase inhibitor cocktails from Sigma-Aldrich?for protein extracts and then performed to detect marker genes expression by Western blotting.3.Frozen section for hematoxylin and eosin?HE?staining and immunofluorescence stainingFrozen common carotid artery sections?5?m thick?were fixed and processed by HE staining.The intimal hyperplasia area was analyzed to determine the neointimal area and media area using Image J software?National Institutes of Health,Bethesda,MD,USA?.For immunofluorescence staining,sections from different groups of WT and Esdn^-/-mice or Cre^+/-Esdn^flox/floxlox/flox and their control littermates Cre^-/-Esdn^flox/floxlox/flox mice were fixed in pre-cooled acetone?-20°C?for 5 min;after three washes in phosphate-buffered saline and a 10 min treatment with 3%H₂O₂,sections were serum-blocked and incubated with antibodies against ESDN,CD31 or SMa-actin.4.Cell culture and treatmentVSMCs were isolated from the thoracic aorta of 8-100g male Sprague-Dawley rat as we reported previously.Briefly,VSMCs were cultured in low glucose Dulbecco's-modified Eagle's medium?DMEM,Life Scientific?supplement with 20%fetal bovine serum?FBS?,100 U/mL penicillin and 100mg/mL streptomycin.VSMCs were maintained at 37?in a humidified atmosphere containing 5%CO₂,and only passage 3-5 cells were used in the following experiments.For ESDN knockdown experiment,VSMCs were seeded in 60-mm tissue culture dishes in antibiotic-free media supplement with 20%FBS for 12-24 hours,then transfected with specific duplex ESDN siRNA or Universal Negative Control?Ribo?using Lipofectamine RNAiMax reagent according to the manufacturer's protocol.After 48 hours,VSMCs were serum starved for another 24 h and then stimulated with PDGF?10ng/mL?for different times?0,5,15 and 30 min?,the RNA was isolated for detection of transfection efficiency and protein for Western blot assay.5.Isolation of membrane subfractionsTo detect the affection of ESDN on PDGFR-? location,VSMCs were serum-starved for 24 h in DMEM following PDGF treatment?10 ng/mL?,and then cytosolic and membrane fractions were prepared using the Kit?Invent?according to the manufacturer's instruments.6.Surface protein isolation and detection of PDGFR-? on cell surfaceTo measure the relative proportions of the surface and internal pools of PDGFR-?,cell surface proteins were covalently labeled using a membrane-impermeantbiotinylationreagent?NHS-SS-biotin;Pierce,Rockford,IL?.All steps were performed at 4°C.Cells were washed 3 times in PBS and then incubated with 0.15 mg/mL sulfo-NHS-SS-biotin?Pierce?in PBS for 10 minutes with rocking.The unreacted biotinylation reagent was quenched by washing once with 1×PBS?137 mM NaCl,2.7 mM KCl,2 mM KH₂PO₄,10 mM Na₂ HPO₄?,and the cells were then washed a further 3 times with PBS.Cells were then solubilized in lysis buffer?50 mM Tris-cl pH 7.5,150 mM NaCl,0.5%Deoxycholate,1%NP-40,0.1%SDS,1 mM EDTA,complete proteinase inhibitor from Roche Applied Sciences and phosphatase inhibitor cocktails from Sigma-Aldrich?.Cell lysates were centrifuged at14000g for 10 minutes at 4°C and a sample?10?L?was taken from the supernatant,whichrepresentedthetotalcellularPDGFR-?.Streptavidin-agarose beads?Upstate Biotechnology,Lake Placid,NY?were added to the remaining supernatant?100?L packed beads per 500?L lysate?and left to tumble at 4°C for 2 hours.Beads were collected by centrifugation at 14000g for 10 seconds at 4°C and supernatant was removed;this sample represents the internal PDGFR-? pool.The beads were then washed 3 times in lysis buffer at 4°C and protein was extracted from the beads by heating at95°C with sodium dodecyl sulfate-polyacrylamide gel electrophoresis?SDS-PAGE?sample buffer;this represents the surface PDGFR-? pool.Equivalent volumes of all 3 samples were resolved by SDS-PAGE and analyzed by Western blotting.Results1 Genotypic identification for smooth muscle cell specific Esdn knockout micePrimers used for genotype of global Esdn knockout,smooth muscle cell specific Esdn knockout Cre^+/-Esdn^flox/floxlox/flox and control littermate Cre^-/-Esdn^flox/floxlox/flox mice.Primers for global Esdn^-/-mice?PCR product size 360 bp?:Forward primer:5'-CAGCCAAACCTCCAGAAAAG-3'Reverse primer:5'-AAGCTTAAGTGATGTGACAG-3'Primers for Esdn^flox/floxlox/flox mice?PCR product size 280bp,wild type 230 bp?Forward primer:5'-CAGCCAAACCTCCAGAAAAG-3'Reverse primer:5'-TCACAAGAGAGAGGGGTGCT-3'Primers for SM22 Cre mice?positive control 324 bp,Cre 100 bp?SM22-Cre Forward primer:5'-GCGGTCTGGCAGTAAAAACTATC-3'SM22-Cre Reverse primer:5'-GTGAAACAGCATTGCTGTCACTT-3'SM22-Cre positive control Forward primer:5'-CTAGGCCACAGAATTGAAAGATCT-3'SM22-Cre positive control Reverse primer:5'-TAGGTGGAAATTCTAGCATCATC-3'Agarose gel electrophoresis results showed the clear bands in the target region,indicating that the genotypic identification correctly,smooth muscle cell-specific Esdn knockout mice constructed successfully.The expression of ESDN was detected by Western blotting,and the results showed that the expression of ESDN was not detected in the vascular tissues of Esdn global knockout mice.Compared with the control mice,the expression of ESDN was detected in the vascular tissues of smooth muscle cell specific knockout mice significantly decreased,which the results further confirmed the genotypic identification is correct.ESDN deletion accelerated neointima formation after ligation.2 ESDN knockout promoted vascular hyperplasia after ligationThe left common carotid arteries from WT and Esdn^-/-mice or Cre^+/-Esdn^flox/floxlox/flox and their control littermates Cre^-/-Esdn^flox/floxlox/flox mice were harvested at different times.Compared with the WT mice,HE staining showed that the intima gradually thickened and the lumen narrowed in aorta of Esdn^-/-mice,and reaching to the peak at 21 days after ligation.Western blotting was used to detect the expression of ESDN on smooth muscle marker protein,and results showed that the expression of OPN?proliferation marker?increased gradually and peaked at 21 d afte liagation in Esdn^-/-mice,while the expression ofSM?-actin and SM22??differentiation marker?gradually decreased in Esdn^-/-mice.Immunofluorescence staining technique was used to observe the effect of ESDN expression on the proliferation of SMC in media area.The results showed that the expression of ESDN co-localizated with the expression of SM?-actin,which indicating that ESDN may affect the proliferation of VSMC and thus affect the process of vascular proliferation.This result was further verified in smooth muscle cell-specific knockout mice.3 ESDN knockdown promoted PDGF signaling pathwayIn order to investigate the molecular mechanism of ESDN deficiency promoting vascular proliferation,rat VSMCs were cultured and ESDN expression was knocked down by siRNA.After serum starvation,VSMCs were treated with PDGF-BB?10 ng/mL?for different time.Western blot was used to detect the PDGFR-? phosphorylation and its downstream signaling pathways.The results showed that down-regulation of ESDN promoted PDGF-induced PDGFR-? phosphorylation and reached its peak at 15 min after stimulation with PDGF.At the same time,PDGF downstream signaling molecules related to migration and proliferation of VSMC,phosphorylation of MAPK-ERK and Akt were also increased.Knockdown of ESDN expression can obviously promoted PDGF signaling pathway,and accelerated the migration and proliferation of VSMC,which is consistent with the results of whole animal.4 ESDN knockdown accelerated PDGF-induced PDGFR-? endocytosisPDGF can bind to its receptor on cell membrane,triggers PDGFR-? phosphorylation and traffics into the cytoplasm.To investigate the ESDN affects the PDGFR-? endocytosis,we used biotin-labeled cell surface proteins,and then streptavidin was used to detect the distribution of PDGFR-?.The results showed that ESDN knockdown significantly reduced PDGFR-? distribution on the plasma membrane,indicating that downregulation of ESDN expression promoted PDGF-induced PDGFR-? endocytosis process;while the distribution of PDGFR-? in the cytoplasm was significantly increased than that of the control group at the same time point,which further verified that downregulation of ESDN promote the PDGF signaling pathway probably through enhancing the PDGFR-? endocytosis process.Conclusions:1 ESDN null mice and smooth muscle specific knockout mice accelerates proliferation of media vascular smooth muscle cells and neointimal hyperplasia;2 Downregulation ESDN expression promotes PDGF signaling pathway;3 ESDN affects the PDGF signaling pathway probably through modulating the PDGFR-? endocytosis process...