Effects And Mechanism Of CCNI On EPCs Function During The Repair Process After Vascular Injury

1. Background and Objective:Percutaneous coronary intervention (PCI) is widely used in the treatment of coronary artery disease. Despite the advent of drug-eluting stent that is promising in reducing the incidence of restenosis, it remains a considerable clinical challenge. Endothelial progenitor cells (EPCs), which can differentiate into mature endothelial cells (ECs), are increasingly recognized to play a key role in vascular regeneration. However, early reendothelialization by regenerated ECs is critical for reducing or preventing postangioplasty restenosis and thrombosis. It is crucial, therefore, to understand the molecular mechanism leading to EPCs differentiation and contribution to the repair process after vascular injury.CCN1, a secreted matricellular protein belonging to the CCN family, is expressed by all types of vascular cells. It is required for vascular development and is a potent regulator of angiogenesis. CCN1-deficient mice undergo embryonic lethality as a result of placental vascular insufficiency and compromised vessel integrity. Recombinant CCN1 induces angiogenesis in vivo, and stimulates ECs survival, migration and tube formation in culture. Moreover, it's noteworthy that expression of CCN1 is up-regulated in vascular disorders, including atherosclerosis, mechanical injury, hypertension, and tumorigenesis. Besides, CCN1 has been associated with tissue self-renewal, such as bone fracture repair, liver regeneration, and cutaneous wound healing, suggesting a role for CCN1 in vascular regeneration, potentially involving circulating EPCs. However, the precise role of CCN1 in vascular regeneration remains largely unexplored. Evidence indicates that CCN1 is critically involved in the regulation of mesenchymal stem cells (MSCs) adhesion, migration, and differentiation. Recent investigation demonstrates that CCN1, in the plasma and endothelium surface, promotes the migration, adhesion, and recruitment of circulating human bone marrow-derived CD34+ progenitor cells to ECs and, thus play an important role in microenvironment-mediated biological properties of EPCs. All of these findings support the hypothesis that CCN1 may have a role in mediating EPCs functions, contributing to the neovascularization and vascular repair process after injury.In this study, we overexpressed CCN1 and siRNA to evaluate the possible role of CCN1 on EPCs proliferation, migration, differentiation, and participation in vascular regeneration. Our findings provide a novel insight into understanding of biological function of CCN1 and the molecular mechanisms behind EPCs-mediated vascular regeneration.2. Methods:2.1 Construction of recombinant adenoviral vectorsAdenoviral vectors repectively expressing CCN1 and Id1 were generated using the AdEasy system. Briefly, full-length rat CCN1 and Id1 cDNA were generated by RT-PCR using total RNA from Sprague–Dawley (SD) rat heart and spleen. The cDNA was first TA-cloned into pMD19-T simple vector and then subcloned into pAdTrack-CMV, resulting in pAdTrack-CCN1 and pAdTrack-Id1. The shuttle vectors were used to generate recombinant adenoviruses according to the manufacturer's protocol. All PCR-amplified fragments and cloning junctions were verified by DNA sequencing and enzymatic digestion. An adenovirus encoding green fluorescent protein (GFP; Ad-GFP) was used as control.2.2 Expression and function of CCN1 during vascular repair following rat carotid artery balloon angioplastyTo evaluate the role of CCN1 in vascular repair in vivo, we first examined the expression of CCN1 in balloon-injured rat carotid artery, using real-time quantitative PCR and Wester blot analysis. Next, adenoviral vectors expressing exogenous CCN1 or GFP as control were constructed and injected into balloon-injured artery to investigate CCN1 gene function during vascular injury. Overexpression of CCN1 was confirmed by immunohistochemistry. No observable adverse side effect (mortality or any other clinical signs of distress/morbidity) was found in experimental animals. Evans Blue dye was administered to evaluate reendothelialization at 4, 7, and 14 days after injury, and the neointimal formation was assessed at 14 and 21 days following vascular injury.2.3 Effecs of CCN1 on EPCs proliferation, migration, and differentiation EPCs were isolated by density gradient centrifugation and cultured in low glucose DMEM supplemented with 10% FCS and 10ng/mL VEGF. To confirm the EPCs phenotype, cells were incubated with DiI-acLDL for 4 hours, fixed with 4% paraformaldehyde and then incubated with FITC-labeled lectin (UEA-1) for 1 hour. Dual-stained cells positive for both DiI-acLDL and UEA-1 were identified as EPCs. Additionally, flow cytometry (FACS) analysis was performed using antibodies against rat CD45, CD133, CD34, and VEGFR-2. To investigate the effect of CCN1 on EPCs migaration, proliferation, and differentiation in vitro, we transduced Ad-CCN1 and pGenesil-CCN1 into EPCs that were cultured in serum- and VEGF- free medium.2.4 Molecular mechanisms underlying CCN1 effects on EPCsTo identify the molecular mechanism that might be involved in the effect of CCN1 on EPCs, we performed microarray analysis on EPCs stimulated with CCN1 or the GFP control, using GEArray. Genes that were either up-regulated or down-regulated 2-fold or more were determined. Moreover, we next examined the effect of CCN1 on Id1 expression, and overexpressed Id1 in EPCs to further confirm the impact of Id1 in CCN1-induced differentiation of EPCs.3. Results:3.1 Recombinant adenoviral vectors expressing CCN1 or Id1Full length cDNA encoding either CCN1 or Id1 was amplified by RT-PCR using total RNA from Sprague–Dawley (SD) rat heart or spleen. The cDNA was first TA-cloned into pMD19-T simple vector and then subcloned into adenoviral shuttle vector pAdTrack-CMV. Recombinant adenovirus Ad-CCN1 and Ad-Id1 were generated and purified according to the manufacturer's protocol. The adenovirus virus titer was about 1.2×1010 -2.8×1011 plaque-forming units per millilitre (pfu /ml), as determined by plaque assay.3.2 Expression and function of CCN1 during vascular repair following rat carotid artery balloon angioplasty3.2.1 Rat carotid balloon-injury modelHistological analysis and H&E staining demonstrated that neointimal formation was initiated at 7days and obviously developed at 21day after balloon-mediated vascular injury in control rats. In addition, after transfection with Ad-CCN1 or Ad-GFP, green fluorescence was detected with vascular tissues under a fluorescence microscope at 48 h post-vascular injury, indicating the efficiency of adenovirus transfection in rat carotid arteries.3.2.2 Expression of CCN1 during vascular repair processCCN1 mRNA expression was detected at low levels in normal uninjured control arteries, whereas following vascular injury CCN1 mRNA level was rapidly enhanced with a peak at 12 h which gradually declined thereafter in 4 days and remained elevated for up to at least 21days. CCN1 protein expression was assessed by Western blotting and was consistently found to be up-regulated. Further, immunohistochemistry showed that CCN1 was detected in the intima, media, and adventitia of local vessels.3.2.3 Effect of Ad-CCN1 on vascular reendothelializationEvans Blue dye was administered to evaluate reendothelialization at 4, 7 and 14 days after injury. Nonendothelialized lesions were marked blue about 100% at injured vessels, whereas the reendothelialized area appeared white at uninjured vessels. At all time points, the reendothelialized area in the Ad-CCN1-infected arteries was significantly larger than that in Ad-GFP infected arteries (p<0.05).3.2.4 Effect of Ad-CCN1 on neointimal formationA marked decrease in the neointimal area and I/M ratio (0.26±0.12 vs 0.58±0.18, p<0.01) was shown in Ad-CCN1-treated rats compared with that of control group at day 14. However, no difference was found at 21 days (1.13±0.21 vs 1.20±0.19, p>0.05), suggesting that CCN1 effectively prevented neointimal hyperplasia at early stage.3.3 Effecs of CCN1 on EPCs proliferation, migration, and differentiation3.3.1 EPCs isolation and characterizationAfter 4-7 days of culture, adherent EPCs were characterized by immunofluorescence and flow cytometry analysis (FACS). The majority of cells (>90%) stained positive for DiI-AcLDL and lectin, and expressed endothelial/stem cell markers, including CD34 (90.28%), VEGFR-2 (74.03 %), and CD133 (93.86 %), but not hematopoietic cell marker CD45 (<10 %.), confirming the cell type of EPCs.3.3.2 EPCs transfectionAdenovirus-mediated CCN1 expression was confirmed by fluorescence, RT-PCR, and Western blot analysis. The transfection efficiency was 60% at 24h post-transfection and 80% at 48-72h post-transfection. Four days after introduction of pGenesil1-CCN1, an approximate 50% of CCN1 expression loss was shown, as measured by Western blot and RT-PCR.3.3.3 Effect of Ad-CCN1 on EPCs proliferationThe proliferation of EPCs was not ehanced significantly by Ad-CCN1. Despite a decrease observed in cells transfected with Ad-CCN1 as compared with Ad-GFP (p<0.05), it has no significant meaning in compare with untransfected control (p>0.05). Our findings suggested that adenovirus maight inibit the proliferation of EPCs.3.3.4 Effect of Ad-CCN1 on EPCs migrationOver expression of exogenous CCN1 extensively improved the migration of EPCs, and in fact, the average migrated cell number of the EPCs increased by approximately 3-fold, from 7.1±1.8 to 6.1±2.8 (* P<0.01) compared to that of the control cells.3.3.5 Effect of Ad-CCN1 on EPCs differentiationAfter Ad-CCN1 infection, a noticeable amount of EPCs transformed into a spindle-or polygonal-shaped appearance, which was less observed in control Ad-GFP transduced EPCs. Additionally, the expression of the progenitor marker CD34 was drastically decreased (13.83±5.23% in Ad-CCN1 group vs 59.79±9.61% in Ad-GFP group), whereas the expression of ECs marker VE-cadherin was increased conversely (85.74±7.11% in Ad-CCN1 group vs 14.04±6.28% in Ad-GFP group). Immunofluorescence study showed that the percentage of DiI-acLDL-positive cells was significantly increased in Ad-CCN1 group (93.5±6.3% vs 61.3±9.2%, p<0.05).3.3.6 Role of siRNA-CCN1After introduction of pGenesil1-CCN1, EPCs exhibited a decrease in cell proliferation, migration and differentiation when compared with negative control siRNA transfected cells or untransfected cells (* p< 0.05).3.4 Molecular mechanisms underlying CCN1 effects on EPCs3.4.1 Microarray analysisWe found that at 48 hours upon Ad-CCN1 stimulation, 8 genes were up-regulated in EPCs compared with Ad-GFP controls. Among these were prominent growth factor and receptor genes such as Vegf-c, Kdr, Igf-1, and Ereg, as well as Tnf, Timp2, Itgav, and Gna13. In contrast, 12 genes were significantly down-regulated in EPCs transfected with Ad-CCN1. These genes include Vegf-b, Tgfα, Tgfβ, Mdk, Ptn, Ifn-α1, Cxcl5, Timp3, Thbs, Plau, Akt1, and Sh2d2a, most of which were corresponding to growth factors, receptors, chemokines, and adhesion molecules, pointing to their potential involvement in the effects of CCN1 on EPCs.3.4.2 Role of transcription factor Id1In response to Ad-CCN1, a decrease of Id1 mRNA expression was detected in EPCs at 48 hours (p<0.05). Transfection with Ad-Id1 alone induced no apparent changes in Dil-acLDL positive cells and expression of cell markers compared with that of Ad-GFP. However, cotransfection with Ad-CCN1 and Ad-Id1 significantly inhibited the Ad-CCN1 induced differentiation of EPCs, p<0.05.4. Conclusions:4.1 CCN1 was dynamically expressed in vascular lesions and found to promote reendothelialization and inhibit neointimal formation at the early stage after vascular injury;4.2 Overexpression of CCN1 stimulated EPCs migration and differentiation and that was reversed by siRNA-mediated silencing of CCN1 expression;4.3 There were 8 genes up-regulated and 12 genes down-regulated in EPCs upon Ad-CCN1 stimulation, suggesting their potential involvement in the mechanism of CCN1 effect on EPCs;4.4 Ad-CCN1 inhibited the expression of Id1 in EPCs, and Id1, at least partly, inhibited CCN1-induced differentiation of EPCs.