Effects Of Inhibitor Of DNA Binding-1(Id1) On Proliferation And Migration Of The EPCs And Involved In The Repair Process After Vascular Injury

1. Background and Objective:Enhancement of reendothelialization is a critical therapeutic option to repair injured blood vessels. Regeneration of injured endothelium has been attributed to the migration and proliferation of neighboring endothelial cells (ECs). Mature ECs are terminally differentiated cells with a low proliferative potential, and their capacity to substitute damaged endothelium is limited. Increasing evidence suggests that circulating endothelial progenitor cells (EPCs), which can home to sites of tissue injury and differentiate into mature ECs and participate in re-endothelialization after vascular injury, may be an endogenous repair mechanism to maintain the integrity of the endothelial monolayer by replacing denuded parts of the artery. EPCs also secrete various cytoprotective or pro-angiogenic factors in a paracrine manner to promote the survival and proliferation of ECs. The physiological and therapeutic significance of EPCs in reendothelialization processes is currently the subject of intensive investigation.Mobilization of EPCs by cytokines, growth factors or drugs; infusion ex-vivo expanded EPCs; and EPC-based gene therapy have been suggested to contribute to re-endothelialization and to inhibit intimal hyperplasia after vascular injury. A critical limitation for therapeutic application of post-natal EPCs is their low number in the circulation (which is even lower in patients with cardiovascular risk factors). The approaches mentioned above have the potential to overcome the problem. The mechanisms underlying reendothelialization by EPCs must be intensively investigated and these mechanisms understood before novel strategies for EPC therapy are translated into clinical applications.During reendothelialization, migration and proliferation of EPCs are the key steps regulated by various mechanisms and signals. The inhibitor of DNA binding 1 (Id1) proteins, an important subfamily member of helix-loop-helix (HLH) transcriptional factors, has been implicated in regulating the growth, proliferation, migration and differentiation of cells. Several studies have focused on the Id1 transcription factor because Id1 knockout mice (Id1+/– Id3–/–) exhibit impaired tumor growth associated with impaired recruitment of EPCs. Restraint of the expression of the cyclin-dependent kinase inhibitor p21 by Id1 is one key element of its activity in facilitating EPC generation in the bone marrow. Recent studies also demonstrated tumor-induced expression of Id1 in EPCs, and conditional Id1 suppression resulted in impaired mobilization of EPCs. These studies shed light on the relationship between Id1 and the functional regulation of EPCs, including recruitment, population and mobilization of EPCs.Based on the available data concerning Id1 on EPC behavior, Id1 may exert potential effects on the migration and proliferation of EPCs, contributing to the neovascularization and vascular repair process after injury. In this study, we overexpressed Id1 and si-RNA to evaluate the possible role of Id1 on EPCs proliferation, migration, and participation in vascular regeneration. Our findings provide a novel insight into understanding of biological function of Id1 and the molecular mechanisms behind EPCs-mediated vascular regeneration.2. Methods:2.1 Construction of recombinant adenoviral vectorsAdenoviral vectors repectively expressing Id1 were generated using the AdEasy system. Briefly, full-length rat 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-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 Effecs of Id1 on spleen derived EPCs proliferation and migration in vitro spleen derived 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 mouse Scal-1 and VEGFR-2. To investigate the effect of Id1 on spleen derived EPCs proliferation and migaration in vitro, we transduced Ad- Id1 and pGenesil- Id1 into EPCs that were cultured in serum- and VEGF- free medium.2.3 Expression and function of Id1 during vascular repair following mice carotid artery injuredTo evaluate the role of Id1 in vascular repair in vivo, firstly, we examined the expression of Id1 in injured mice carotid artery, using PCR and Westen blot analysis. Secondly, we transduced Ad- Id1 and pGenesil- Id1 into EPCs. Thirdly, these transduced EPCs were injected by intravenous tail vein after induction of arterial injury. The injured segments were isolated 14 days after EPCs transplantation. Overexpression of Id1 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 14 day after injury, and the neointimal formation was assessed at 14 day following vascular injury.3. Results:3.1 Recombinant adenoviral vectors expressing Id1Full length cDNA encoding 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-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 Effecs of Id1 on spleen derived EPCs proliferation and migration in vitro3.2.1 spleen derived 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 Scal-1 (83.5%) and VEGFR-2 (57.6 %) confirming the cell type of EPCs.3.2.2 Expression and location of Id1 in EPCsId1 was present at fairly low levels in quiescent EPCs, but was rapidly upregulated upon stimulation with serum and VEGF (a strong growth factor of EPCs) and was detected via mRNA in RT-PCR or by protein in western blotting. To analyze the subcellular localization of Id1, EPCs were fixed and subjected to DAB staining with anti-Id1 multiclonal antibody by immunocytochemical staining: Id1 was localized predominantly in the cytoplasm.3.2.3 EPCs transfectionAdenovirus-mediated Id1 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-Id1, an approximate 60% of Id1 expression loss was shown, as measured by Western blot and RT-PCR.3.2.4 Effect of Ad- Id1 on EPCs proliferationThe proliferation of EPCs was not ehanced significantly by Ad- Id1. Despite a decrease observed in cells transfected with Ad- Id1 as compared with Ad-GFP (p<0.05), it has significant meaning in compare with untransfected control (p<0.05).3.2.5 Effect of Ad- Id1 on EPCs migrationOver expression of exogenous Id1 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.3.2.6 Role of siRNA- Id1After introduction of si-RNA- Id1 EPCs exhibited a decrease in cell proliferation and migration when compared with negative control siRNA transfected cells or untransfected cells (* p< 0.05).3.3 Expression and function of Id1 during vascular repair following mice carotid artery injured3.3.1 Mice carotid injury model Histological analysis and H&E staining demonstrated that neointimal formation was initiated at 7days and obviously developed at 28 day after vascular injury in control mouse. In addition, after transfection with Ad- Id1 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 mice carotid arteries.3.3.2 Expression of Id1 during vascular repair processId1 mRNA expression was detected at low levels in normal uninjured control arteries, whereas following vascular injury Id1 mRNA level was rapidly enhanced with a peak at 14 d which gradually declined thereafter in 14 days and remained elevated for up to at least 28days. Id1 protein expression was assessed by Western blotting and was consistently found to be up-regulated. Further, immunohistochemistry showed that Id1 was detected in the intima and media of local vessels.3.3.3 Effect of Ad- Id1-EPCs transplantation on vascular reendothelializationEvans Blue dye was administered to evaluate reendothelialization at 14 days after injury. Nonendothelialized lesions were marked blue about 100% at injured vessels, whereas the reendothelialized area appeared white at uninjured vessels. The reendothelialized area in the Ad- Id1-EPCs infected arteries was significantly larger than that in Ad-GFP-EPCs, and non-infected groups (reendothelialized area /totle vessel injured area ratio was 68.36±4.51, 43.1±6.59 and 40.5±7.82, respectively, p<0.05), suggesting that transplantation of Ad-Id1-EPCs effectively promoted reendothelialization after vascular injury.3.3.4 Effect of Ad- Id1-EPCs transplantation on neointimal formationNo marked decrease in the I/M ratio was shown in Ad- Id1-EPCs group compared with Ad-GFP-EPCs or non-infected group at day 14 (1.08±0.1, 1.16±0.14 and 1.15±0.17, respectively, p>0.05).4. Conclusions:4.1 Id1 was present at fairly low levels in quiescent EPCs and was localized mainly in the cytoplasm. Id1 was rapidly upregulated upon stimulation with serum and VEGF;4.2 Overexpression of Id1 stimulated spleen derived EPCs proliferation and migration, and that was reversed by si-RNA-mediated silencing of Id1 expression;4.3 Id1 was dynamically expressed in vascular lesions, and attained the peak expression in14 day after injure.4.4 Ad-Id1-EPCs transplantation could home into the vascular injury site and accelerate reendothelialization of denuded vessel, howere, could not inhibit the neointima formation at the same period after vascular injury.