Targeted Dipeptidyl-peptidase IV (CD26)-mediated Bone Marrow-derived Endothelial Precursor Cells (EPCs) Inhibits Laser-induced Choroidal Neovascularization

Background Age-related macular degeneration (AMD) is one of the mostcommon irreversible causes of severe loss of vision, including legal blindness, in theelderly population. Choroidal neovascularization (CNV) is the most severe form of AMD.There are no current treatments that can “cure” AMD or reverse its course, including laserphotocoagulation, verteporfin photodynamic therapy (PDT) and intravitreal injections ofcorticosteroids and anti-angiogenic agents. Generally, these therapies seek to avoid furthervision loss rather than to improve existing vision. Selective targeting of bonemarrow–derived cells (BMCs) has been heralded as a promising avenue for age-relatedmacular degeneration (AMD) therapeutics. Cell therapy using BMCs by ex vivo isolation and local delivery could provide beneficial effects for repair and regeneration of injuredand degenerated retina and choroid tissues. Although BMC therapy with may be the mostexciting avenue for AMD therapeutics, a major barrier to transferring the use ofautologous BMCs into clinical practice is the limited quantity of BMCs in the peripheralcirculation. Technology has not yet reached a stage where ex vivo–expanded BMCs can beroutinely used for cell therapy.Bone marrow–derived endothelial progenitor cells (EPCs) may represent animportant endogenous repair mechanism. In vivo mobilization and expansion of EPCscould supply sufficient autologous cells for tissue repair, thereby circumventing theexisting issues of cells therapy. The interaction of stromal cell-derived factor1α (SDF-1α)and CXCR4has been identified as a principal axis in the regulation of EPC mobilization.A recent finding has shown that SDF-1α degradation and inactivation within the bonemarrow by the peptidase CD26(dipeptidyl peptidase IV, DPP IV) may ultimately result inthe abrogation of the SDF-1/CXCR4axis, which is an important regulatory mechanism formobilization of EPCs. Moreover, CD26/dipeptidylpeptidase IV (DPP IV) modification byangiotensin-converting enzyme (ACE) inhibitor plays a critical role in mobilizing EPCsfrom bone marrow. In spite of bone marrow-derived EPCs have been shown to contributeto CNV, whether the beneficial effect of induced EPCs mobilization has yet to be fullydetermined. In addition, whether homing of EPCs to the site requiring repair may exertspositive effectsf for laser-induced CNV remains to be answered.Objectives The purpose of the present study was to investigate the role of theincreased EPC mobilization by modulation CD26/DPP IV system in the development ofCNV. To determine whether the endogenous repair mechanism of EPCs may beassociated with reactive astrocytes in the laser-induced CNV.This study, using a murine model of laser-induced CNV,(1) observe that imidapril, anACE inhibitor (ACEI), regulate the activity of CD26/DPP IV system in vivo andinvestigate the effects of ACEI on proteolytic activation between bone marrow andperipheral circulation under laser-induced injure conditions;(2) observe that ACEImodulate the function of SDF-1and influence the level of EPC mobilization throughmanipulation of the CD26system in the development of CNV and confirm the underlyingmechanism under injury stress conditions;(3) observe mobilization of EPCs target astrocytes at the retina nerve filber layer (NFL) to intervene development of CNV. Weinvestigate that EPCs target activated astrocytes by manipulation of endogenous repairmechanisms in the laser-induced CNV.Methods CNV in C57BL/6J mice was generated by focal rupture of Bruch’smembrane with a532-nm diode laser. Animals were randomized to5treatment groups.Animals were pretreated intragastrically with phosphate-buffered saline (PBS),intragastrically with imidapril (ACEI) and/or subcutaneously with diprotin-A (a DPP IVantagonist) for days5before photocoagulation and the treatments were continued dailyuntil days14after laser induction. Normal control group is nontreated and nonlasered.(1)Fluorescence-activated cell sorting (FACS) Analysis: Peripheral blood and bone marrowsamples were obtained from all groups after laser-induced CNV day12. Blood was wasobtained by cardiac puncture, while bone marrow cells were extracted from femurscirculating cells and bone marrow cells were identified using a nucleated cell fraction. Thequantity of CD26+cells in the bone marrow and peripheral blood was estimated using afluorescein isocyanate (FITC)-conjugated anti-mouse CD26antibody. The nucleated cellswere double labelled with FITC-conjugated anti-CD34monoclonal antibody andphycoerythrin (PE)-conjugated anti-Flk-1antibody. Circulating EPCs were quantified byenumerating CD34+and VEGFR2+cells. The cells were examined by flow cytometry.(2)SDF-1filamentous actin (F-actin) polymerization of lymphocytes by is quantified bymeans of flow cytometry. Whole blood samples collected from the animals afterlaser-induced CNV day12. The cells were stimulated with or without30nM of SDF-1and stained with FITC phalloidin. The intracellular fluorescence was determined by FACSanalysis. The lymphocyte population was gated, and median fluorescence was measured.(3) Blood samples were obtained by via a tail vein on pretreated with different drug dailyon days1,5and after laser-induced CNV day3, day7, day14. The total number of whiteblood cells (WBCs) was enumerated with a Neubauer hematocytometer.(4) CD26proteolytic activity was examined by a microplate reader. plasma and bone marrowextracellular fluids were obtained after laser-induced CNV day12. Proteolytic activity wasdetermined by measuring the amount of p-Nitroaniline (pNA) formed and the DPP IVactivity in units/liter (U/L) calculated in the supernatant at405nm.(5) Enzyme linkedimmunosorbent assay (ELISA) measurement of cytokines: peripheral blood murine samples and bone marrow were obtained from all groups after laser-induced CNV day3,7,14. Blood was centrifuged to collect plasma, while bone marrow extracellular fluids wereextracted from femurs as previously described. Plasma and bone marrow SDF-1αconcentrations were measured with a mouse SDF-1α ELISA kit.(6) Fluoresceinangiography (FA) was performed after laser photocoagulation day13and fluoresceinleakage was evaluated. Late-stage FA images were taken at4-6minutes post injection. Thegrading protocol used to compare leakage in experimentally induced CNV.(7)Histopathology study evaluated CNV Lesion size by hematoxylin-eosin (HE) staining andfluorescein isocyanate-Griffonia simplicifolia isolectin-B4(FITC-isolectin B4) Stainingafter laser-induced CNV day14.(8) Glial fibrillary acidic protein (GFAP) immunoreactivecells were identified as astrocytes cells by immunofluorescence labelling within the retinaNFL after laser-induced CNV day14.(9) The protein expression of GFAP was measuredin the retinal by western blotting after laser-induced CNV day14.Results (1) ACEI disrupted the balance of the proteolytic activity of bone marrowand peripheral blood by manipulation of CD26/DPP IV system. In the bone marrow,imidapril was primarily through the upregulation of CD26/DPP IV activity on bonemarrow cell rather than through altering the number of CD26+cells after laser-inducedCNV day12. In the blood, imidapril significantly decreased CD26+cell numbers, leadingto a decrease in total CD26/DPP IV activity, because decrease the number of WBCsthrough an anti-inflammatory effect. Furthermore, Diprotin A can completely blockedCD26/DPP IV activity caused by imidapril intervention.(2) ACEI has the ability tonegatively regulate the SDF-1/CXCR4axis by manipulation of CD26/DPP IV system.Imidapril-treated animals after laser-induced CNV demonstrated significant increases inplasma–SDF-1compared with other groups after laser-induced CNV day12. Meanwhile,SDF-1concentrations in the bone marrow were significantly lower in the imidapril groupcompared to the other groups. Imidapril also caused a significant decrease SDF-1inducedactin polymerization in whole blood. These phenomena were associated with a lowerCD26activity in bone marrow but higher in the blood in the imidapril group, compared tothe other groups. The inversing SDF-1concentration gradient between the bone marrowand the peripheral blood significantly mobilized EPCs from the bone marrow into thecirculation. The effect of imidapril on EPC mobilization in laser-inducd CNV was significantly blocked by Diprotin A.(3) The mobilization of EPC significantly inhibitedthe laser-induced CNV and reactive astrocytes. FA was conducted after laserphotocoagulation day13. Treatment with imidapril significantly decreased CNV leakagecompared to the other groups after laser-induced CNV. HE and FITC-Isolectin B4-stainedalso showed mice treated with imidapril suppressed CNV volume versus other groups.GFAP up-regulation is a hallmark of reactive astrocytes. Immunofluorescence taining ofGFAP showed mice treated with imidapril GFAP immunoreactive cell were significantlyincreased in the retina NFL versus the other groups. Imidapril group was significantlyincreased the protein expression of GFAP compare with other groups after thelaser-induced CNV day14.Conclusions (1) ACE inhibitor effectively regulated the activity of CD26/DPPIV system in laser-induced CNV. In the bone marrow, imidapril did not alter CD26+cellnumbers; however, it did amplify CD26/DPP IV activity. In the blood, through theanti-inflammatory effect, imidapril significantly decreased CD26+cell numbers, leading toa decrease in total CD26/DPP IV activity.(2) CD26/DPP IV has the ability to negativelyregulate the SDF-1/CXCR4axis by proteolytic degradation of SDF-1α in bone marrow,which significantly reduced the concentration of SDF-1α and increases in plasma–SDF-1α.The inversing SDF-1α concentration gradient between the bone marrow and the peripheralblood significantly mobilized EPCs from the bone marrow into the circulation.(3) Themobilization of bone marrow-derived EPCs target reactive astrocytes at the retina NFLandinhibits the laser-induced CNV in term of manipulation of endogenous repair avenue.Bone marrow-derived EPCs mobilization can inhibit the laser-induced CNV. Thebeneficial effect of induced EPCs mobilization in the development of CNV wasdetermined firstly. CD26/DPP IV modification by ACEI activate EPCs mobilization mayrepresent an important endogenous repair avenue. The therapeutic strategy may providethe novel target of EPC mobilization to treat CNV and overcome the existing problems ofBMC-based therapy in clinical application. Present and future therapies will be aimed atmodifying the course of CNV development. EPC mobilization may provide a novelavenue of therapeutic exploitation to promote repair and re-generation，then it may betimely to consider therapies other than antiangiogenesis.