Effects Of PDGFR-β Over-expression In EPCs On The Repair Process After Vascular Injury

1. Background and Objective:Endothelial injury is an important pathophysiological basis of atherosclerosis andrestenosis after percutaneous coronary intervention (PCI).Endothelial repair andregeneration is therefore a crucial step in treating cardiovascular diseases.Althoughendothelial cells (ECs) repair and regeneration are thought to rely only on the adjacentmature ECs.However, advances in therapeutics are greatly limited by the insufficient denovo differentiation of pre-existing mature ECs.Endothelial progenitor cells (EPCs) are a special type of bone marrow-derivedprogenitor cells, which can enter into circulation, proliferation and differentiate intovascular ECs, but not yet express phenotype of mature ECs, nor form vascular precursors.Itincludes multiple stages of cells from the mother cells to mature ECs. Early EPCs whichcan repair the blood vessels, and the late EPCs for its strong proliferation ability, play rolesin angiogenesis.Previous many basic and clinical studies have showed that EPCs can berecruited into the sites of injury, differentiate into ECs, replace dysfunctionalendothelium,and play important roles in postnatal neovascularization, maintaining normalendothelial function and endothelial repairing after vascular injury.Platelet-Derived Growth Factor (PDGF) was initially isolated in platelets. Thecomplete PDGF family consists of proteins derived from four polypeptides A, B, C, and D,that form four disulfide-linked homodimers (PDGF-AA,-BB,-CC,-DD) and oneheterodimer (PDGF-AB).These five PDGF dimers bind to PDGFR-α,-β,and-αβ withdifferent affinities. PDGF–BB is a good mitogen in vitro and can act as a chemotacticfactor in vivo. It is the only PDGF dimer that binds to all three PDGFR isoforms: PDGFR-α,PDGFR-β, and PDGFR-αβ. The fact that PDGF-BB or PDGFR-β has been implicated in the pathogenesis of vascular proliferative disorders such as atherosclerosis and restenosis afterangioplasty. Evidence suggests that the facilitative role of PDGF-BB or PDGFR-β inmediating arterial intimal hyperplasia after in the formation of atherosclerotic lesions and anangioplasty. Interestingly, targeting PDGF-BB with an anti-PDGF-BB antibody or usinginhibitors of PDGF-BB and PDGFR-β signal transduction blocked intimal thickening inanimal models.Previous studies have indicated that the interaction between PDGF-BB and PDGFR-βis the key for the proliferation and migration of cells. We note that PDGF-BB andPDGFR-β interaction induces the phosphorylation of PDGFR and activatesphosphoinositide3-kinase (PI3K).Moreover, the PI3K signaling pathway also contributes toa number of cell processes, including cell proliferation, survival, motility, andangiogenesis.EPCs play important roles in the regeneration of the vascular endothelial cells.Basedon the available data concerning PDGF-BB and PDGFR-β on cell biological function,PDGF-BB and PDGFR-β may exert potential effects on the proliferation, migration, andangiogenesis of EPCs, contributing to the neovascularization and vascular repair processafter injury. Therefore, in this study, we overexpressed PDGFR-β to evaluate the possiblerole of PDGF-BB on EPCs proliferation, migration and angiogenesis. In addition, weinjected PDGFR-β transfected EPCs into the sites of vascular injury to observeEPCs-mediated vascular regeneration. Our findings may be provided a novel insight for thetreatment of vascular injury.2. Methods:2.1Endogenous PDGFR-β expression in spleen-derived EPCs2.1.1EPCs culture and characterizationBriefly, Spleen-derived mononuclear cells (MNCs) were isolated using densitygradient centrifugation with Lymphoprep. After three times rinses, cells were plated inhuman fibronectin-coated cell culture flasks and propagated in Dulbecco's Modified EagleMedium: nutrient mixture F-12(DMEM/F-12) culture medium supplemented with20%fetal calf serum (FCS),10ng/ml recombinant human vascular endothelial growth factor(VEGF). For characterization, cell growth and morphology were observed. To observe thefunction of EPCs, uptake of acetylated LDL and UEA-1binding were carried out. Additionally, analysis of cultured spleen-derived MNCs for Sca-1(a stem/progenitor cellmarker) and VEGFR-2(an endothelial cell marker) by fluorescence activated cell sorting(FACS).2.1.2PDGFR-β localization and expression in EPCs2.1.2.1The localization of the endogenous PDGFR-β in EPCs was investigated byimmunofluorescence2.1.2.2The mRNA and protein expression level of PDGFR-β in EPCs was examinedby Semi-quantitative RT-PCR and Western blot2.2Gene transfection of spleen-derived EPCs2.2.1Liposome-mediated cell transfectionAfter EPCs were cultured for10or11days and reached60-70%confluence,transfection was performed using a Lipofectamine2000reagent according to theinstruction manual. The DNA (μg) to Lipofectamine2000(μl) ratio was1:2.2.2.2To estimate the transfection efficiencyIn order to evaluate the transfection efficiency, the number of EGFP-positive cells wasdivided by the total number of EPCs in the same area.2.2.3The mRNA and protein expression level of PDGFR-β in EPCs was examined bySemi-quantitative RT-PCR and Western blot.2.3Secretion of PDGF-BB by EPCsTo compare the expression level of PDGF-BB between the untransfected and transfectedEPCs, we measured the concentration of PDGF-BB in the supernatant of the culture mediumusing Enzyme-Linked Immunosorbent Assay (ELISA).2.4Effects of PDGFR-β overexpression on PDGF-BB-induced EPC biologicalfunctions in vitroTo observe the effects of exogenous PDGFR-β transfection and differentconcentrations of PDGF-BB stimulation on the biological functions of EPCs, the threegroups of EPCs from PDGFR-β transfection (the control group, the pEGFP-N2group, andthe pEGFP-N2-PDGFR-β group) were treated with PDGF-BB of different concentrations (0,10,20,40,80, or160ng/ml).2.4.1Effects of PDGFR-β overexpression on PDGF-BB-induced EPC proliferationThe proliferation of EPCs was examined by the colorimetric MTS assay. 2.4.2Effects of PDGFR-β overexpression on PDGF-BB-induced EPC migrationEPC migration was assayed using a Transwell system containing8μm polycarbonatefilter inserts in24-well plates.2.4.3Effects of PDGFR-β overexpression on PDGF-BB-induced EPC tube-formationEPC tube-like formation was evaluated with the In Vitro Angiogenesis Assay Kit.2.5The role of PI3K/Akt signal pathway in PDGF-BB-induced EPCs biologicalfunctionsTo examine whether the PDGFR-β/PI3K signaling pathway is involved inPDGF-BB-induced biological functions of EPCs, the two groups of EPCs (the control groupand the pEGFP-N2-PDGFR-β group) were pretreated with AG1295(a PDGFR kinaseinhibitor), LY294002(a PI3K inhibitor), and sc-221226(an Akt inhibitor),respectively, for1h before treated with PDGF-BB at maximal effective concentration.2.5.1EPC proliferation assayThe proliferation of EPCs was assayed by the colorimetric MTS assay.2.5.2EPC migration assayEPC migration was examined using a Transwell system containing8μm polycarbonatefilter.2.5.3EPC tube-formation assayEPC tube-like formation was evaluated with the In Vitro Angiogenesis Assay Kit.2.5.4The phosphorylation levels of PDGFR-β, PI3K, and Akt were examined bywestern blotTo investigate whether the PDGFR-β/PI3K/Akt signaling pathway was involved inEPCs, the phosphorylation levels of PDGFR-β, PI3K, and Akt were examined by westernblot after exposure to PDGF-BB, AG1295, LY294002, and sc-221226.2.6Function of PDGFR-β overexpression in EPCs during vascular repair2.6.1The mouse model of arterial injury2.6.2PDGF-BB localization and expression in injured vessels2.6.2.1The localization of the endogenous PDGF-BB in injured vessels wasinvestigated by immunofluorescence2.6.2.2The mRNA and protein expression level of PDGF-BB in injured vessels wasexamined by real-time PCR and Western blot 2.6.3Spleen-derived EPCs transplantationInjury of carotid artery was preformed in splenectomized mice.Mice receivedenhanced green fluorescent protein(EGFP)-labeled EPCs or PDGFR-β-EPCs by tail veininjection directly after endothelial injury of the carotid artery and again24h later.2.6.4EPCs tracingTo observe whether transfused EPCs were able to homing to the site of injury andpositive of vWF confirmed the endothelial phenotype,images were taken by laser scanning.2.6.5Effect of EPCs transplantation on vascular reendothelializationEvans blue staining was performed to evaluate reendothelialization at7days after injury.2.6.6Effect of EPCs transplantation on neointimal formationTo assess neointimal thickness, HE-stained cryosections was performed at14days afterinjury.2.6.7Effect of EPCs transplantation on medial apoptotic cellsMedial apoptotic cells were detected by immunostaining for TUNEL using in situ celldeath detection kit at7days after injury. TUNEL labeling index calculated by dividingpositive labeled cells by total cell number.3. Results:3.1Endogenous PDGFR-β expression in spleen-derived EPCs3.1.1Characterization of spleen-derived EPCs3.1.1.1After cultured for4-7days, spleen-derived MNCs exhibited a spindle-shaped,endothelial cell-like morphology or formed cluster-like structures. After10days, cells formedcord-like structures. After20days, cells became confluent, and looked like cobblestones.3.1.1.2Spleen-derived MNCs showed uptake of acetylated LDL and UEA-I bindingafter4-7days in culture. Most adherent cells were LDL and UEA-I double positive (91.2±1.8%).3.1.1.3FACS analysis of cultured spleen-derived MNCs,71.7±0.93%of these cellsexpressed mouse stem-cell marker Sca-1, and52.49±9.27%expressed endothelial cellmarker VEGFR-2.3.1.2PDGFR-β localization and expression in EPCs3.1.2.1The subcellular localization of PDGFR-β in EPCsThe localization of the endogenous PDGFR-β in EPCs was investigated by immunofluorescence after cultured for7days.PDGFR-β was found to be localized in theplasma membrane of the cells (88.6±4.3%).3.1.2.2The mRNA and protein expression level of PDGFR-β in EPCs was examined bySemi-quantitative RT-PCR and Western blotThe expression of PDGFR-β in EPCs progressively increased with differentiation timeas shown by semi-quantitative RT-PCR and Western blot using cells from day4,7,14, and21. PDGFR-β was present at fairly low levels in EPCs.3.2Gene transfection of spleen-derived EPCs3.2.121h after transfection, EGFP expression was detected by laser scanning confocalmicroscopy in transfected EPCs, while no EGFP was detected in EPCs.3.2.2The transfection efficiency was about50-60%.3.2.372h post-transfection, the level of PDGFR-β in the pEGFP-N2-PDGFR-β groupwas significantly increased compared to the control group or the pEGFP-N2group, asshown by semi-quantitative RT-PCR and Western blot (P <0.01).3.3Secretion of PDGF-BB by EPCsPDGF-BB is a secretory protein, and the concentration of PDGF-BB in the supernatantof culture medium was17.2±2.0pg/ml. The concentration of PDGF-BB was significantlylower in the pEGFP-N2-PDGFR-β group than in the control or pEGFP-N2group at48h and72h post-transfection(P＜0.01).3.4Effects of PDGFR-β overexpression on PDGF-BB-induced EPC biologicalfunctions in vitro3.4.1Effects of PDGFR-β overexpression on PDGF-BB-induced EPC proliferationThe maximum proliferation induced by PDGF-BB occurred at20ng/ml in both thecontrol and pEGFP-N2groups and at80ng/ml in the pEGFP-N2-PDGFR-β group.3.4.2Effects of PDGFR-β overexpression on PDGF-BB-induced EPC migrationThe maximum migration induced by recombinant PDGF-BB occurred at20ng/ml inboth the control and pEGFP-N2groups and at80ng/ml in the pEGFP-N2-PDGFR-β group.3.4.3Effects of PDGFR-β overexpression on PDGF-BB-induced EPC tube-formationThe maximum tube formation induced by recombinant PDGF-BB occurred at20ng/mlfor both the control and pEGFP-N2groups, and at80ng/ml for the pEGFP-N2-PDGFR-βgroup. 3.5The role of PI3K/Akt signal pathway in PDGF-BB-induced EPCs biologicalfunctions3.5.1EPC proliferation assayThe PDGF-BB-induced proliferation in the control group (by20ng/ml PDGF-BB) andthe pEGFP-N2-PDGFR-β group (by80ng/ml PDGF-BB) were all significantly inhibited bythe pretreatment of AG1295, LY294002, or sc-221226.The result indicated that thePI3K/Akt signaling pathway participated in the PDGF-BB-induced proliferation of EPCs.3.5.2EPC migration assayThe PDGF-BB-induced migration in the control group (by20ng/ml PDGF-BB) andthe pEGFP-N2-PDGFR-β group (by80ng/ml PDGF-BB) were all significantly inhibited bythe pretreatment of AG1295, LY294002, or sc-221226.The result indicated that thePI3K/Akt signaling pathway participated in the PDGF-BB-induced migration of EPCs.3.5.3EPC tube-formation assayThe PDGF-BB-induced angiogenesis in the control group (by20ng/ml PDGF-BB)and the pEGFP-N2-PDGFR-β group (by80ng/ml PDGF-BB) were all significantlyinhibited by the pretreatment of AG1295, LY294002, or sc-221226.The result indicatedthat the PI3K/Akt signaling pathway participated in the PDGF-BB-induced angiogenesis ofEPCs.3.5.4The phosphorylation levels of PDGFR-β, PI3K, and Akt were examined bywestern blotPretreatment with AG1295, LY294002, or sc-221226attenuates PDGF-BB stimulatedphosphorylation of PDGFR-β, PI3K, and Akt.3.6Function of PDGFR-β overexpression in EPCs during vascular repair3.6.1The mouse model of carotid artery injuryHE staining demonstrated that neointimal formation was initiated at7days andobviously developed at28days after carotid artery injuryin control mice.3.6.2PDGF-BB localization and expression in injured vessels3.6.2.1The localization of the PDGF-BB in injured vessels was investigated byimmunofluorescence showed that PDGF-BB was observed in the intima, media of localinjured carotids, whereas rarely been observed in uninjured carotids.3.6.2.2The mRNA and protein expression level of PDGF-BB in injured vessels was examined by real-time PCR and Western blot PDGF-BB mRNA and protein expression wasfound at low levels in uninjured carotids. No significant change in PDGF-BB mRNA andprotein expression was detected in4days, whereas following vascular injury PDGF-BBmRNA and protein level was rapidly enhanced with a peak at7days which graduallydeclined to normal thereafter in21days.3.6.3EPCs tracing24hours after EPCs transplantation, EGFP-expressing cells were found at the injurysite by laser scanning.10days after EPCs transplantation, cells were found in injured sitesthat were positive of EC marker vWF too.3.6.4Effect of PDGFR-β overexpressed EPCs on vascular reendothelializationAfter injury of the carotid artery, reendothelialized area appeared white at uninjuredvessels,whereas the nonendothelialized lesions were marked blue about100%at injuredvessels by Evans blue staining.7days after injury, the reendothelialized area in thePDGFR-β-EPCs group was significantly larger than that in EGFP-EPCs group (p<0.05).Our data showed that the transplantation of PDGFR-β over-expressed EPCs can promotereendothelialization in the early phase after carotid artery injury.3.6.5Effect of PDGFR-β overexpressed EPCs on neointimal formationA significantly decrease in the neointimal area and I/M ratio was shown inPDGFR-β-EPCs group compared with that of EGFP-EPCs group at day14(p<0.05). Ourresults indicated that the transplantation of PDGFR-β over-expressed EPCs can inhibitneointimal formation after carotid artery injury.3.6.6Effect of EPCs transplantation on medial apoptotic cells7days after injury, TUNEL labeling index was significantly greater in thePDGFR-β-EPCs group than EGFP-EPCs group (p<0.01). Our data showed that thetransplantation of PDGFR-β over-expressed EPCs can promote medial cells apoptosis in theearly phase after carotid artery injury.4. Conclusions:4.1EPCs are resident in spleen, which can differentiate into ECs in specific condition.4.2PDGFR-β was present at fairly low levels in spleen-derived EPCs and waslocalized mainly in the plasma membrane.4.3PDGF-BB induces EPCs proliferation, migration and angiogenesis. 4.4Over-expression of PDGFR-β promotes PDGF-BB-induced proliferation,migration, and angiogenesis of EPCs through PI3K/Akt signaling pathway.4.5PDGFR-β over-expressed EPCs significantly promote reendothelialization in theearly phase after mouse carotid artery injury.4.6PDGFR-β over-expressed EPCs inhibit neointimal formation after mouse carotidartery injury.4.7PDGFR-β over-expressed EPCs significantly promote medial cells apoptosis in theearly phase after mouse carotid artery injury.