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PLGA Microspheres
Journal Pre-proof Neovascularization by sustained delivery of G-CSF, EPO and VEGF using dextran/ PLGA microspheres Chong Dong Liu, Xiao Fei Tu, Feng Chen PII:
S0890-5096(19)30864-7
DOI:
https://doi.org/10.1016/j.avsg.2019.10.033
Reference:
AVSG 4682
To appear in:
Annals of Vascular Surgery
Received Date: 17 August 2019 Revised Date:
23 September 2019
Accepted Date: 1 October 2019
Please cite this article as: Liu CD, Tu XF, Chen F, Neovascularization by sustained delivery of G-CSF, EPO and VEGF using dextran/PLGA microspheres, Annals of Vascular Surgery (2019), doi: https:// doi.org/10.1016/j.avsg.2019.10.033. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.
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Neovascularization by sustained delivery of G-CSF, EPO and VEGF using
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dextran/PLGA microspheres
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Chong Dong Liua, #, Xiao Fei Tub, #, Feng Chena,*
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a
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Nanchang 330006, China
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b
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China,
Department of Vascular Surgery, the first affiliated Hospital, Nanchang University,
Department of General Surgery, Jiangxi Provincial Children's Hospital, Nanchang 330006,
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# These authors contributed equally to this work.
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* Corresponding author: Feng Chen, Department of Vascular Surgery, the first affiliated
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Hospital, Nanchang University, Nanchang 330006, China. E-mail [email protected]
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KEYWORDS
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Granulocyte-colony stimulating factor, Erythropoietin, Vascular endothelial growth factor,
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Sustained release, Microsphere, Ischemia
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ABSTRACT
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Introduction
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Therapeutic neovascularization has some obstacles, such as it requires more than one
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pro-angiogenic factor, and these factors have short half-lives. To overcome these obstacles,
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combined delivery of granulocyte-colony stimulating factor (G-CSF), erythropoietin (EPO)
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and vascular endothelial growth factor (VEGF) using protein/dextran/poly(lactic-co-glycolic
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acid) (PLGA) sustained-release microspheres was proposed to promote neovascularization.
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Methods
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Dextran microparticles loaded with G-CSF, EPO or VEGF were prepared and encapsulated in
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PLGA microspheres to obtain protein-dextran-PLGA microspheres. The release behavior of
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microspheres was studied in vitro. The protein/dextran/PLGA microspheres were injected
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into the ischemic hindlimbs of rats. Neovascularization in ischemic muscle was measured.
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Results
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Microspheres released G-CSF, EPO and VEGF in vitro for more than 4 weeks. Combined
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therapy with VEGF, EPO and G-CSF promoted the expression of B-cell lymphoma-2 and
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stromal cell-derived factor 1, cellular proliferation and the incorporation of C-X-C chemokine
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receptor 4 positive cells. Capillary density and smooth muscle α-actin+ vessel density were
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higher in the combined treatment of VEGF, EPO and G-CSF than in the single factor
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treatment.
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Conclusion
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The combined and sustained delivery of VEGF, EPO and G-CSF using dextran-PLGA
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microspheres had a more significant neovascularization effect than monotherapy with each
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45
factor alone. This combined therapy might be a promising treatment for ischemic vascular
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diseases.
47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
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67
Introduction
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Ischemic vascular disease is the main cause of morbidity and mortality in the world [1].
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Therapeutic neovascularization is a promising treatment for ischemic vascular diseases. It can
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be achieved by delivering the related pro-angiogenic growth factors, genes or cells to
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stimulate neovascularization [2, 3].Therapeutic neovascularization based on angiogenic
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factors has the following obstacles: (1) neovascularization is a complex process, requiring
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more than one type of pro-angiogenic growth factor or cell; (2) most pro-angiogenic growth
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factors have short half-lives.
75
In this study, vascular endothelial growth factor (VEGF) combined with erythropoietin
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(EPO) and granulocyte-colony stimulating factor (G-CSF) were used as the therapeutic
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growth factors to compensate for the deficiency of single growth factor. VEGF induces
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angiogenesis by promoting endothelial cells (ECs) proliferation and migration. EPO and
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G-CSF are hematopoietic growth factors, which are commonly used to mobilize
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hematopoietic stem cells and progenitor cells. They promote neovascularization by boosting
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the mobilization, proliferation and differentiation of multiple cell populations through
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arteriogenesis and vasculogenesis mechanisms [4-8].
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The half-life of most pro-angiogenic factors is very short. In order to maintain the
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effective concentration for therapeutic neovascularization, frequent injections are needed.
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Sustained-release drug delivery system provides an excellent alternative. The most popular
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delivery system is microsphere system made of poly(lactic-co-glycolic acid) (PLGA). It has
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inherent advantages of biocompatibility and biodegradability. However, during the
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encapsulation process, protein aggregation or denaturation is prone to occur due to water/oil
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and water/air interfacial tension [9, 10].These aggregated or denatured proteins can evoke
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immunogenicity and lose therapeutic activity [11, 12].In order to overcome these
91
shortcomings, some methods have been developed [10, 13-15].For example, an ideal method
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is to pre-load protein into the dextran microparticles and then microencapsulate
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protein-dextran microparticles into PLGA microspheres.
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In this study, we hypothesized that combination therapy with G-CSF, EPO and VEGF
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using the protein/dextran/PLGA microsphere delivery system could promote
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neovascularization, and was more effective than sigle factor therapy in a rat hindlimb
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ischemia model. VEGF/dextran/PLGA, G-CSF/dextran/PLGA and EPO/dextran/PLGA
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microspheres were prepared. The morphology and in vitro release characteristics of
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microspheres were studies. Fibrin gel simultaneously loaded with these three types of
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microspheres was injected into the muscle tissue of the hindlimb ischemia model in rats, and
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neovascularization in ischemic tissue was detected.
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Methods
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Production of protein/dextran/PLGA microspheres
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As previously described [14, 16], protein/dextran/PLGA microspheres were prepared. Human
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G-CSF, EPO or rat VEGF 165 (PeproTech), dextran (Sigma-Aldrich), and PEG
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(Sigma-Aldrich) were mixed at a mass ratio of 1:5:50, stirred for 1 min at 1500 rpm,
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refrigerated for 12 h at -80
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washed three times with dichloromethane. The final protein-dextran microparticles were
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collected and evaporated for 24 h, then suspended in the PLGA (15%, w/w) (Sigma-Aldrich)
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solution dissolved in dichloromethane, stirred immediately for 60 s, emulsified into NaCl
, and then lyophilized for 24 h. The lyophilized powder was
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(5%, w/w) solution added with polyvinyl alcohol (1%, w/w) (Sigma-Aldrich), and stirred at
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2000 rpm. Initial protein/dextran/PLGA microspheres were obtained. In order to solidify the
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protein/dextran/PLGA microspheres, the above microspheres emulsion was added to NaCl
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(10%, w/w) solution at 4
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VEGF/dextran/PLGA, G-CSF/dextran/PLGA and EPO/dextran/PLGA microspheres were
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washed with pure water, and then lyophilized for 24 h.
, and stirred for 4 h at 150 rpm. Finally, the hardened
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Morphology of protein/dextran/PLGA microspheres
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The morphological characteristics of G-CSF/dextran/PLGA, EPO/dextran/PLGA and
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VEGF/dextran/PLGA microspheres were observed by scanning electron microscopy (SEM).
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Before image scanning, gold was deposited on the surface of microspheres by vapor
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deposition.
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Encapsulation efficiency of protein/dextran/PLGA microspheres
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G-CSF/dextran/PLGA, EPO/dextran/PLGA and VEGF/dextran/PLGA microspheres were
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dissolved in dichloromethane respectively, and centrifuged at 10,000 rpm for 5 min. The
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supernatant was removed. The insoluble pellets were collected and dissolved in phosphate
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buffer saline (PBS). VEGF, G-CSF and EPO ELISA kits (R&D Systems) were used to
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determine the concentration of released factors. The encapsulation efficiency of
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protein/dextran/PLGA microspheres was evaluated by the following formula: encapsulation
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efficiency =Wa/Wt×100%. In this formula, Wa refers to the actual weight of protein
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microencapsulated into dextran/PLGA microspheres, and Wt refers to the theoretical weight
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of protein microencapsulated into dextran/PLGA microspheres.
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Release profile of protein/dextran/PLGA microspheres
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The 20mg of G-CSF/dextran/PLGA, EPO/dextran/PLGA and VEGF/dextran/PLGA
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microspheres were suspended in 2mL PBS, respectively, and continuously oscillated at 37°C.
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The released PBS was collected and the same amount of fresh PBS was added every two days.
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VEGF, G-CSF and EPO ELISA kits (R&D Systems) were used to determine the contents of
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G-CSF, EPO and VEGF. For each type of microsphere, release analysis was repeated six
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times. The cumulative release amount of proteins was plotted against time for each sampling
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interval time to get the cumulative release profiles.
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Rat ischemic hindlimb model and grouping
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Sprague-Dawley rats (male, weight 200g) were anesthetized by intraperitoneal injection of 60
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mg/kg ketamine. From the inguinal ligament to the knee, a vertical longitudinal incision was
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made in the left hindlimb. The femoral artery and its branches were dissected, ligated and
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completely resected. Fibrin gel was prepared by mixing 2 ml of the thrombin component (400
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IU/mL), 2 ml of the sealer protein containing fibrinogen, factor XIII and aprotinin, and
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protein/dextran/PLGA microspheres. One day after operation, 60 rats were randomly divided
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into five groups (12 in each group): G+E+V, in which the rats were treated with intramuscular
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injection of fibrin gel loaded with G-CSF/dextran/PLGA, EPO/dextran/PLGA and
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VEGF/dextran/PLGA microspheres in the anterior tibialis muscle (volume = 200µl,
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G-CSF=70 µg/rat, EPO=2000U/rat, VEGF=70 µg/rat); V, in which the rats got treatment with
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fibrin gel loaded with VEGF/dextran/PLGA microspheres; G, in which the rats got treatment
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with fibrin gel loaded with G-CSF/dextran/PLGA microspheres; E, in which the rats got
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treatment with fibrin gel loaded with EPO/dextran/PLGA microspheres; C, as a control group,
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in which the rats got treatment with fibrin gel loaded with empty dextran/PLGA microspheres.
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The dose of growth factors was based on previous similar studies [4, 5, 8, 17-21]. The
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protocols for all experiments involving animal surgery were approved by the institutional
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animal care and use committee of Nanchang University.
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Western blot, histological and immunohistochemical analysis
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On day 7 and 28 post-treatment, tibialis anterior muscle specimens were harvested and split
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into two parts for use in both Western blot and histological analysis. Muscle tissue was cut
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into small pieces, grinded using a homogenizer with lysis buffer containing 1 mmol/L DTT,
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400 µmol/L PMSF, 20 mmol/L Hepes (pH 7.4), 1 mmol/L Na3VO4, 2 mmol/L EDTA, 10%
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glycerol, 1% Triton X-100, 2 µmol/L leupeptin and 10 units/mL aprotinin (Sigma), and
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incubated at 4°C for 30 min. Lysates were centrifuged at 10,000 rpm at 4°C for 10 min and
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the supernatant was collected. Protein concentration was determined by BCA method. The 20
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ug protein extract from each sample was heated at 95°C for 10 min in a loading buffer,
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separated onto 10% SDS-PAGE, transferred to 0.22 µm PVDF membranes (Millipore),
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blocked in 5% non-fat milk for 2 hours at room temperature, and incubated overnight at 4 °C
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with the respective primary antibodies: rabbit antibodies against stromal cell derived factor 1
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(SDF-1) (Abcam), B-cell lymphoma-2 (Bcl-2) (Abcam), β-actin (Abcam), Bcl-2 associated x
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protein (Bax) (Abcam) and C-X-C chemokine receptor 4 (CXCR4) (Abcam). After washing
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three times in TBST, the membranes were incubated at room temperature with a
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HRP-conjugated goat anti-rabbit secondary antibody (Vector Laboratories) for 1 h followed
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by washing three times in TBST, and the protein bands were visualized using a commercial
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ECL kit (Amersham) and Kodak film. The protein bands of SDF-1, Bcl-2, Bax, CXCR4 and
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β-actin were quantitatively analyzed using ImageJ Software.
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Muscle tissue was embedded in OCT compound and snap-frozen. They were cut into
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serial sections 6 µm thick. In order to detect capillary ECs, the sections were stained with
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alkaline phosphatase. In order to detect smooth muscle α-actin (SMA) positive vessels,
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immunohistochemical staining was performed with a rabbit antibody against SMA (Abcam).
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The stained capillaries and SMA+ vessels on sections were quantitatively analyzed using
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ImageJ Software. And immunohistochemical staining with primary antibodies against SDF-1,
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Bcl-2, CXCR4 and proliferating cell nuclear antigen (PCNA) (Abcam) were performed on
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sections. Non-immune IgG of the corresponding species was used as negative control to
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confirm the specificity of primary antibody. The sections were stained according to the
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manufacturer’s protocols.
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Statistics
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SPSS 13.0 (SPSS Inc, Chicago) was used to analyze the data which were presented as
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means±S.E. Comparison of means was achieved using Student’s t-test and one-way ANOVA
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with Tukey’s honestly significant differences test. The significance was assigned at P <0.05.
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Results
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Morphological characteristics of protein/dextran/PLGA microspheres
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The morphological characteristics of the protein/dextran/PLGA microspheres loaded with
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G-CSF, EPO or VEGF were evaluated with SEM scanning method. Protein/dextran/PLGA
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microspheres were spherical with smooth surface and diameters of 40-120 µm (Figure 1).
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Encapsulation efficiency of protein/dextran/PLGA microspheres
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ELISA kits were used to evaluate the protein encapsulation efficiency of
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protein/dextran/PLGA microspheres. The protein encapsulation efficiencies of
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G-CSF/dextran/PLGA, EPO/dextran/PLGA and VEGF/dextran/PLGA microspheres were
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84%±4.2, 85%±4.0, 82%±3.8 (w/w), respectively.
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Release profile of protein/dextran/PLGA microspheres
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Protein/dextran/PLGA microspheres released angiogenic factors for more than 4 weeks (Fig.
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1). The cumulative releases of G-CSF, EPO and VEGF were approximately 89.5%, 90.3%
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and 91.2% respectively, and the amounts of burst release were about 23.5%, 25.8% and
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26.5% respectively during the first two days.
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Neovascularization in ischemic muscle
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To detect capillary ECs, alkaline phosphatase staining was performed on ischemic muscle
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sections. As shown in Fig. 2 and Fig. 3, the capillary density (number/muscle fiber) was
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significantly higher in G, E, V and G+E+V groups than in control group (p
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capillary density in G+E+V group was the highest.
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0.05), and the
At post-treatment day 28, to detect SMA+ vessels, immunohistochemical analysis with
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an antibody against SMA were performed on ischemic muscle sections. As showed Fig. 2 and
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Fig. 3, the density of SMA positive vessels in G, V and G+E+V groups was significantly
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higher than that in the control group (p
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group was the highest.
0.05), and the density of SMA+ vessels in G+E+V
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Enhanced Bcl-2 expression and cellular proliferation
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To detect the expression of Bcl-2, at post-treatment day 7, immunohistochemical analysis and
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Western blot analysis were performed using antibodies against Bcl-2 and Bax. As shown in
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Fig. 4 and Fig. 5, it seemed that G-CSF, EPO and VEGF all could promote the expression of
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Bcl-2. The Bcl-2 expression in group G+E+V was significantly higher than that in other
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groups (p
0.05).
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To examine cellular proliferative activity, immunohistochemical analysis with an
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antibody against PCNA was performed. In Fig. 4, it seemed that G-CSF, EPO and VEGF all
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seemed to promote cellular proliferation in varying degrees. The cellular proliferative activity
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in G+E+V group was higher than that in other groups.
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Enhanced SDF-1 expression and CXCR4+ cells incorporation
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To examine the SDF-1 expression, at post-treatment day 7, immunohistochemical analysis
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and Western blot analysis with an antibody against SDF-1 were performed. As shown in Fig.
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6 and Fig. 7, it seemed that EPO alone promoted the expression of SDF-1 in group E. The
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expression of SDF-1 in group G+E+V was significantly higher than that in other groups (p
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0.05).
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In Fig. 2, muscle tissues with HE staining showed varying degrees of cellular infiltration
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in each group. In order to detect the incorporation of CXCR4+ cells in ischemic tissues,
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immunohistochemical analysis and Western blot analysis using an antibody against CXCR4
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were performed. As shown in Fig. 6 and Fig. 7, it was found that combination therapy with
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G-CSF, EPO and VEGF could promote the incorporation of CXCR4+ cells more effectively
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than single factor therapy.
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Discussion
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An important obstacle to growth factor-based therapeutic neovascularization is the short
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half-life of pro-angiogenic growth factors. To solve this problem, a variety of sustained drug
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release systems have been developed to control the release of pro-angiogenic factors and
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protect them from degradation. PLGA microspheres are the most widely used [17, 22], and
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firstly approved by the Food and Drug Administration of the United States. However, during
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the microencapsulation process of PLGA microspheres, growth factors tend to denaturate or
259
aggregate due to the effect of water/oil or water/air interfacial tension [9, 10]. In order to
260
avoid these defects, an effective method is to pre-load proteins into dextran microparticles,
261
and then microencapsulate these protein/dextran microparticles into PLGA microspheres by a
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solid-in-oil-in-water method [10, 13-15]. It was found that these protein/dextran/PLGA
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microspheres produced by solid-in-oil-in-water method had higher encapsulation efficiency,
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lower initial burst release, longer release period and better bioactivity preservation than
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protein-PLGA microspheres produced by water-in-oil-in-water method. In this study, on the
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basis of the combination of PLGA microspheres and protein/dextran microparticles,
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VEGF/dextran/PLGA, G-CSF/dextran/PLGA and EPO/dextran/PLGA microspheres were
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produced. The results showed that more than 80% of drug-loaded growth factors could be
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released from microspheres efficiently over 4 weeks in vitro. In the rat hindlimb ischemia
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model, it was also found that the released VEGF, G-CSF and EPO all could significantly
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promote neovascularization by increasing the density of capillaries or SMA+ vessels. As
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shown in our results, protein/dextran/PLGA microspheres sustained release system was an
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ideal drug delivery system for combinational therapeutic neovascularization.
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In this study, it was found that VEGF, G-CSF and EPO all significantly increased the
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expression of Bcl-2, and the number of Bcl-2+/PCNA+ proliferating cells in the ischemic
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muscle. First of all, VEGF, as a mitogen to ECs, can inhibit the apoptosis of ECs and
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stimulate the proliferation of ECs [17, 19, 23]. VEGF can promote the expression of Bcl-2 in
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ECs and increase the density of PCNA+ ECs. Secondly, G-CSF can facilitate mobilization,
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recruitment and survival of bone marrow and peripheral bold derived leukocytes, which can
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express pro-angiogenic factors such as VEGF and PDGF to promote neovascularization [6,
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18, 24]. Therefore, G-CSF can also promote the expression of Bcl-2 and PCNA in leukocytes
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and other effector cells [21]. Thirdly, EPO is a hematopoietic cytokine, which can promote
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the proliferation, differentiation and mobilization of erythroid progenitor cells. EPO receptor
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(EPOR) is expressed not only in erythroid progenitor cells, but also in all sorts of
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non-hematopoietic cells, such as ECs [7, 20]. EPO/EPOR can activate the signal transduction
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proteins such as Janus kinase 2, signal transducer and activator of transcription which can
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control cellular proliferation, survival and gene expression [7, 20]. EPO/EPOR can enhance
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the proliferation, survival and migration of ECs, protect them from ischemia and apoptosis,
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and promote angiogenesis in vitro and in vivo [4, 7, 20, 25]. Similarly, EPO could also
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enhance the expression of Bcl-2 and PNCA in ECs. G-CSF, VEGF and EPO all could
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increase the number of Bcl-2+/PCNA+ expression cells in ischemic muscle, thus protecting
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cells from apoptosis, promoting cell proliferation, and inducing angiogenesis. Compared with
293
monotherapy with G-CSF, EPO or VEGF alone, combined treatment with G-CSF, EPO and
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VEGF could significantly promote the expression of Bcl-2 and PCNA.
295
In this study, it was found that EPO/dextran/PLGA microspheres could significantly
296
promote SDF-1 expression and increase the incorporation of CXCR4+ cells in ischemic
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tissues. Lin et al. [26] also found that the increased SDF-1 expression was mediated by EPO,
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since treatment with an antibody against EPOR markedly blocked this effect. It seemed that
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EPO directly affected the expression of SDF-1. Other studies found that EPO induced the
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release of SDF-1 from hematopoietic cells, particularly platelets, and contributed to
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neovascularization of ischemic limb [5, 27, 28]. CXCR4, as a main SDF-1 receptor, is widely
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and constitutively expressed by bone marrow derived cells [28]. CXCR4 positive
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pro-angiogenic cells include hematopoietic cells, endothelial progenitor cells, smooth muscle
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progenitor cells and pericytes. They all have the ability to promote neovascularization
305
directly or indirectly [29]. SDF-1/CXCR4 can regulate some key aspects of blood vessel
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development, stem cells recruitment and postnatal vasculogenesis [29-32]. It was concluded
307
that EPO could play an avtive role in neovascularization by increasing the expression of
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SDF-1.
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VEGF receptor 2 (VEGFR2) and CXCR4 positive cells represent specific subgrouops of
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pro-angiogenic cells, have overlapping phenotypes, and are mobilized by VEGF-A and
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SDF-1, respectively [28]. VEGF-A can promote angiogenesis and bone marrow cell
15
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recruitment by activating SDF-1–CXCR4 pathway [19, 28]. EPO/EPOR plays an important
313
role in angiogenesis induced by ischemia through the upregulation of VEGF/VEGFR system
314
which promotes the recruitment of endothelial progenitor cells and other bone marrow
315
derived proangiogenic cells, and enhances neovascularization in ischemic tissue [6, 28]. In
316
summary, EPO and VEGF seem to have synergistic effects in mobilizing and recruiting
317
CXCR4+ cells to promote neovascularization in ischemic tissues [28].
318
G-CSF is a hematopoietic growth factor that promotes mobilization and proliferation of
319
bone marrow derived hematopoietic stem cells and progenitor cells. By mobilizing and
320
stimulating bone marrow-derived monocytes and progenitor cells to secrete various
321
angiogenic factors or differentiate into endothelial cells, smooth muscle cells or pericytes,
322
G-CSF can also promote the angiogenesis, arteriogenesis and vasculogenesis [6, 18, 24].
323
Through the CXCR4/SDF-1 axis, G-CSF could mobilize and recruitt CXCR4+ cells into
324
ischemic tissue to promote neovascularization in ischemic tissues.
325
The above mechanisms are summarized as follows. Firstly, G-CSF can mobilize
326
hematopoietic cells and progenitor cells, including CXCR4+ cells from bone marrow.
327
Secondly, as a source of SDF-1, these mobilized hematopoietic cells can increase the
328
expression of SDF-1 activated by EPO. More SDF-1 expression can recruit more CXCR4+
329
cells. Thirdly, because of the overlapping phenotypes of VEGFR2+ and CXCR4+ in some
330
cells, VEGF can also recruit CXCR4+ cells. Taking these effects together, G-CSF, EPO and
331
VEGF have synergistic effects on the SDF-1 expression, mobilization and recruitment of
332
CXCR4 positive cells which are beneficial to neovascularization. In this study, it was found
333
that combined treatment with G-CSF, EPO and VEGF had stronger effects on the expression
16
334
of SDF-1, and incorporation of CXCR4+ cells in ischemic tissues than monotherapy with
335
VEGF, EPO or G-CSF alone. Combined therapy could achieve better neovascularization
336
effect than monotherapy.
337
One limitation of this study was the deficiency of more in-depth mechanistic study and
338
discussion. Another limitation was the lack of evaluation of systemic toxicity and side effects.
339
Still another limitation was that this study utilized a small animal model. Further studies were
340
needed to repeat these experiments in large animal models, and assess the clinical potential of
341
this treatment.
342 343
Conclusion
344
The release of G-CSF, EPO and VEGF from protein/dextran/PLGA microspheres lasted for
345
more than 4 weeks in vitro. Combined therapy with VEGF, EPO and G-CSF using
346
dextran/PLGA microspheres had synergistic effects in promoting the expression of Bcl-2 and
347
cellular proliferation, increasing SDF-1 expression and CXCR4+ cells incorporation in
348
ischemic muscle. Compared with monotherapy with VEGF, EPO or G-CSF alone, this
349
combined therapy had more significant neovascularization effect. It may be a promising
350
treatment for ischemic vascular diseases such as cardiac ischemia, limb ischemia, and wound
351
healing.
352 353
Source of Funding
354
This work was supported by grants from the National Natural Science Foundation of China
355
(81460083, 81860094), Natural Science Foundation of Jiangxi Province (20142BAB215034,
17
356
20141BBG70032, 20152ACB21026).
357 358
Compliance with Ethical Standards
359
Conflicts of Interest
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conflict of interest in relation to this article.
361
Ethical Approval
362
the care and use of animals were followed.
All authors have approved the final article and declare no potential
All applicable international, national, and/or institutional guidelines for
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References 1. Tierney, E.F., Gregg, E.W., Narayan, K.M. Leading causes of death in the United States. Journal of the American Medical Association 2006;295:383; author reply 384. 2. Cao, Y., Hong, A., Schulten, H., et al. Update on therapeutic neovascularization. Cardiovascular Research 2005;65:639-648. 3. Lassaletta, A.D., Chu, L.M., Sellke, F.W. Therapeutic neovascularization for coronary disease: current state and future prospects. Basic research in cardiology 2011;106:897-909. 4. Kobayashi, H., Minatoguchi, S., Yasuda, S., et al. Post-infarct treatment with an erythropoietin-gelatin hydrogel drug delivery system for cardiac repair. Cardiovascular Research 2008;79:611-620. 5. Kato, S., Amano, H., Ito, Y., et al. Effect of erythropoietin on angiogenesis with the increased adhesion of platelets to the microvessels in the hind-limb ischemia model in mice. Journal of Pharmacological Sciences 2010;112:167-175. 6. Spadaccio, C., Rainer, A., Trombetta, M., et al. A G-CSF functionalized scaffold for stem cells seeding: a differentiating device for cardiac purposes. Journal of Cellular and Molecular Medicine 2011;15:1096-1108. 7. Kimáková, P., Solár, P., Solárová, Z., et al. Erythropoietin and Its Angiogenic Activity. International Journal of Molecular Sciences 2017;18:1519. 8. Klopsch, C., Skorska, A., Ludwig, M., et al. Intramyocardial angiogenetic stem cells and epicardial erythropoietin save the acute ischemic heart. Disease Models & Mechanisms 2018;11(6). 9. Schwendeman, S.P. Recent advances in the stabilization of proteins encapsulated in injectable PLGA delivery systems. Critical Reviews in Therapeutic Drug Carrier Systems 2002;19:73-98. 10. Jin, T., Zhu, J., Wu, F., et al. Preparing polymer-based sustained-release systems without exposing proteins to water-oil or water-air interfaces and cross-linking reagents. Journal of Controlled Release: official journal of the Controlled Release Society 2008;128:50-59.
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11. Jiang, W., Schwendeman, S.P. Stabilization and controlled release of bovine serum albumin encapsulated in poly(D, L-lactide) and poly(ethylene glycol) microsphere blends. Pharmaceutical Research 2001;18:878-885. 12. Maas, C., Hermeling, S., Bouma, B., et al. A role for protein misfolding in immunogenicity of biopharmaceuticals. The Journal of Biological Chemistry 2007;282:2229-2236. 13. Govardhan, C., Khalaf, N., Jung, C.W., et al. Novel long-acting crystal formulation of human growth hormone. Pharmaceutical Research 2005;22:1461-1470. 14. Geng, Y., Yuan, W., Wu, F., et al. Formulating erythropoietin-loaded sustained-release PLGA microspheres without protein aggregation. Journal of Controlled Release: official journal of the Controlled Release Society 2008;130:259-265. 15. Yuan, W., Geng, Y., Wu, F., et al. Preparation of polysaccharide glassy microparticles with stabilization of proteins. International Journal of Pharmaceutics 2009;366:154-159. 16. Zhang, Z.D., Xu, Y.Q., Chen, F., et al. Sustained delivery of vascular endothelial growth factor using a dextran/poly(lactic-co-glycolic acid)-combined microsphere system for therapeutic neovascularization. Heart and Vessels 2019;34:167-176. 17. Cleland, J.L., Duenas, E.T., Park, A., et al. Development of poly-(D,L-lactide--coglycolide) microsphere formulations containing recombinant human vascular endothelial growth factor to promote local angiogenesis. Journal of Controlled Release: official journal of the Controlled Release Society 2001;72:13-24. 18. Deindl, E., Zaruba, M.M., Brunner, S., et al. G-CSF administration after myocardial infarction in mice attenuates late ischemic cardiomyopathy by enhanced arteriogenesis. FASEB Journal : official publication of the Federation of American Societies for Experimental Biology 2006;20:956-958. 19. Grunewald, M., Avraham, I., Dor, Y., et al. VEGF-induced adult neovascularization: recruitment, retention, and role of accessory cells. Cell 2006;124:175-189. 20. Janmaat, M.L., Heerkens, J.L., de Bruin, A.M., et al. Erythropoietin accelerates smooth muscle cell-rich vascular lesion formation in mice through endothelial cell activation involving enhanced PDGF-BB release. Blood 2010;115:1453-1460. 21. Liu, S.P., Lee, S.D., Lee, H.T., et al. Granulocyte colony-stimulating factor activating HIF-1alpha acts synergistically with erythropoietin to promote tissue plasticity. PLoS One 2010;5:e10093. 22. Soppimath, K.S., Aminabhavi, T.M., Kulkarni, A.R., et al. Biodegradable polymeric nanoparticles as drug delivery devices. Journal of Controlled Release : official journal of the Controlled Release Society 2001;70:1-20. 23. Sato, K., Wu, T., Laham, R.J., et al. Efficacy of intracoronary or intravenous VEGF165 in a pig model of chronic myocardial ischemia. Journal of the American College of Cardiology 2001;37:616-623. 24. Misao, Y., Takemura, G., Arai, M., et al. Importance of recruitment of bone marrow-derived CXCR4+ cells in post-infarct cardiac repair mediated by G-CSF. Cardiovascular research 2006;71:455-465. 25. Noguchi, C.T., Wang, L., Rogers, H.M., et al. Survival and proliferative roles of erythropoietin beyond the erythroid lineage. Expert reviews in molecular medicine 2008;10:e36.
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26. Lin, J.S., Chen, Y.S., Chiang, H.S., et al. Hypoxic preconditioning protects rat hearts against ischaemia-reperfusion injury: role of erythropoietin on progenitor cell mobilization. The Journal of physiology 2008;586:5757-5769. 27. Jin, D.K., Shido, K., Kopp, H.G., et al. Cytokine-mediated deployment of SDF-1 induces revascularization through recruitment of CXCR4+ hemangiocytes. Nature medicine 2006;12:557-567. 28. Petit, I., Jin, D., Rafii, S. The SDF-1-CXCR4 signaling pathway: a molecular hub modulating neo-angiogenesis. Trends in immunology 2007;28:299-307. 29. Kucia, M., Ratajczak, J., Ratajczak, M.Z. Bone marrow as a source of circulating CXCR4+ tissue-committed stem cells. Biology of the Cell 2005;97:133-146. 30. Ceradini, D.J., Kulkarni, A.R., Callaghan, M.J., et al. Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1. Nature Medicine 2004;10:858-864. 31. De Falco, E., Porcelli, D., Torella, A.R., et al. SDF-1 involvement in endothelial phenotype and ischemia-induced recruitment of bone marrow progenitor cells. Blood 2004;104:3472-3482. 32. Seeger, F.H., Rasper, T., Koyanagi, M., et al. CXCR4 expression determines functional activity of bone marrow-derived mononuclear cells for therapeutic neovascularization in acute ischemia. Arteriosclerosis, thrombosis, and vascular biology 2009;29:1802-1809.
20
468 469 470 471 472 473 474
Figure legends
475
Figure 1 A, A scanning electron micrograph of protein/dextran/PLGA microspheres. B, In
476
vitro release profiles of pro-angiogenic growth factors from protein/dextran/PLGA
477
microspheres.
478
Figure 2 Representative microscopic photographs of alkaline phosphatase staining to detect
479
capillary (AP), immunostain with an antibody against smooth muscle α-actin to detect SMA+
480
vessels (α-SMA), and staining with hematoxylin and eosin (HE). C group received empty
481
dextran/PLGA microspheres as control group; G group received G-CSF/dextran/PLGA
482
microspheres; E group received EPO/dextran/PLGA microspheres; V group received
483
VEGF/dextran/PLGA microspheres; G+E+V group three types of microspheres.
484
Figure 3 A, Densities of capillary in ischemic muscle obtained from the rats at post-treatment
485
day 7 and day 28. B, Densities of α-SMA+ vessel at post-treatment day 28. C group received
486
empty dextran/PLGA microspheres as control group; G group received G-CSF/dextran/PLGA
487
microspheres; E group received EPO/dextran/PLGA microspheres; V group received
488
VEGF/dextran/PLGA microspheres; G+E+V group three types of microspheres.
489
Quantification of capillary density as number of capillaries per fiber on the tissue sections.
21
490
Quantification of α-SMA positive vessels density as number of SMA+ vessels per fiber. *
491
p<0.05 vs. group C, # p<0.05 vs. group G+E+V.
492
Figure 4 Representative microscopic photographs of immunostaining with antibodies against
493
Bcl-2 and PCNA. C group received empty dextran/PLGA microspheres as control group; G
494
group received G-CSF/dextran/PLGA microspheres; E group received EPO/dextran/PLGA
495
microspheres; V group received VEGF/dextran/PLGA microspheres; G+E+V group three
496
types of microspheres.
497
Figure 5 Western blotting showing the expression of Bcl-2 and PCNA. Quantification of
498
protein expression in bar graphs. Expression was normalized to loading control GAPDH, and
499
expressed relative to its corresponding GAPDH. C group received empty dextran/PLGA
500
microspheres as control group; G group received G-CSF/dextran/PLGA microspheres; E
501
group received EPO/dextran/PLGA microspheres; V group received VEGF/dextran/PLGA
502
microspheres; G+E+V group three types of microspheres. * p<0.05 vs. group C, # p<0.05 vs.
503
group G+E+V.
504
Figure 6 Representative microscopic photographs of immunostaining with antibodies against
505
SDF-1 and CXCR4. C group received empty dextran/PLGA microspheres as control group;
506
G group received G-CSF/dextran/PLGA microspheres; E group received EPO/dextran/PLGA
507
microspheres; V group received VEGF/dextran/PLGA microspheres; G+E+V group three
508
types of microspheres.
509
Figure 7 Western blotting showing the expression of SDF-1 and CXCR4. Quantification of
510
protein expression in bar graphs. Expression was normalized to loading control GAPDH, and
511
expressed relative to its corresponding GAPDH. C group received empty dextran/PLGA
22
512
microspheres as control group; G group received G-CSF/dextran/PLGA microspheres; E
513
group received EPO/dextran/PLGA microspheres; V group received VEGF/dextran/PLGA
514
microspheres; G+E+V group three types of microspheres. * p<0.05 vs. group C, # p<0.05 vs.
515
group G+E+V.
S0890-5096(19)30864-7
DOI:
https://doi.org/10.1016/j.avsg.2019.10.033
Reference:
AVSG 4682
To appear in:
Annals of Vascular Surgery
Received Date: 17 August 2019 Revised Date:
23 September 2019
Accepted Date: 1 October 2019
Please cite this article as: Liu CD, Tu XF, Chen F, Neovascularization by sustained delivery of G-CSF, EPO and VEGF using dextran/PLGA microspheres, Annals of Vascular Surgery (2019), doi: https:// doi.org/10.1016/j.avsg.2019.10.033. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.
1
1
Neovascularization by sustained delivery of G-CSF, EPO and VEGF using
2
dextran/PLGA microspheres
3
Chong Dong Liua, #, Xiao Fei Tub, #, Feng Chena,*
4 5 6
a
7
Nanchang 330006, China
8
b
9
China,
Department of Vascular Surgery, the first affiliated Hospital, Nanchang University,
Department of General Surgery, Jiangxi Provincial Children's Hospital, Nanchang 330006,
10
# These authors contributed equally to this work.
11
* Corresponding author: Feng Chen, Department of Vascular Surgery, the first affiliated
12
Hospital, Nanchang University, Nanchang 330006, China. E-mail [email protected]
13 14 15 16 17
KEYWORDS
18
Granulocyte-colony stimulating factor, Erythropoietin, Vascular endothelial growth factor,
19
Sustained release, Microsphere, Ischemia
20 21 22
2
23
ABSTRACT
24
Introduction
25
Therapeutic neovascularization has some obstacles, such as it requires more than one
26
pro-angiogenic factor, and these factors have short half-lives. To overcome these obstacles,
27
combined delivery of granulocyte-colony stimulating factor (G-CSF), erythropoietin (EPO)
28
and vascular endothelial growth factor (VEGF) using protein/dextran/poly(lactic-co-glycolic
29
acid) (PLGA) sustained-release microspheres was proposed to promote neovascularization.
30
Methods
31
Dextran microparticles loaded with G-CSF, EPO or VEGF were prepared and encapsulated in
32
PLGA microspheres to obtain protein-dextran-PLGA microspheres. The release behavior of
33
microspheres was studied in vitro. The protein/dextran/PLGA microspheres were injected
34
into the ischemic hindlimbs of rats. Neovascularization in ischemic muscle was measured.
35
Results
36
Microspheres released G-CSF, EPO and VEGF in vitro for more than 4 weeks. Combined
37
therapy with VEGF, EPO and G-CSF promoted the expression of B-cell lymphoma-2 and
38
stromal cell-derived factor 1, cellular proliferation and the incorporation of C-X-C chemokine
39
receptor 4 positive cells. Capillary density and smooth muscle α-actin+ vessel density were
40
higher in the combined treatment of VEGF, EPO and G-CSF than in the single factor
41
treatment.
42
Conclusion
43
The combined and sustained delivery of VEGF, EPO and G-CSF using dextran-PLGA
44
microspheres had a more significant neovascularization effect than monotherapy with each
3
45
factor alone. This combined therapy might be a promising treatment for ischemic vascular
46
diseases.
47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
4
67
Introduction
68
Ischemic vascular disease is the main cause of morbidity and mortality in the world [1].
69
Therapeutic neovascularization is a promising treatment for ischemic vascular diseases. It can
70
be achieved by delivering the related pro-angiogenic growth factors, genes or cells to
71
stimulate neovascularization [2, 3].Therapeutic neovascularization based on angiogenic
72
factors has the following obstacles: (1) neovascularization is a complex process, requiring
73
more than one type of pro-angiogenic growth factor or cell; (2) most pro-angiogenic growth
74
factors have short half-lives.
75
In this study, vascular endothelial growth factor (VEGF) combined with erythropoietin
76
(EPO) and granulocyte-colony stimulating factor (G-CSF) were used as the therapeutic
77
growth factors to compensate for the deficiency of single growth factor. VEGF induces
78
angiogenesis by promoting endothelial cells (ECs) proliferation and migration. EPO and
79
G-CSF are hematopoietic growth factors, which are commonly used to mobilize
80
hematopoietic stem cells and progenitor cells. They promote neovascularization by boosting
81
the mobilization, proliferation and differentiation of multiple cell populations through
82
arteriogenesis and vasculogenesis mechanisms [4-8].
83
The half-life of most pro-angiogenic factors is very short. In order to maintain the
84
effective concentration for therapeutic neovascularization, frequent injections are needed.
85
Sustained-release drug delivery system provides an excellent alternative. The most popular
86
delivery system is microsphere system made of poly(lactic-co-glycolic acid) (PLGA). It has
87
inherent advantages of biocompatibility and biodegradability. However, during the
88
encapsulation process, protein aggregation or denaturation is prone to occur due to water/oil
5
89
and water/air interfacial tension [9, 10].These aggregated or denatured proteins can evoke
90
immunogenicity and lose therapeutic activity [11, 12].In order to overcome these
91
shortcomings, some methods have been developed [10, 13-15].For example, an ideal method
92
is to pre-load protein into the dextran microparticles and then microencapsulate
93
protein-dextran microparticles into PLGA microspheres.
94
In this study, we hypothesized that combination therapy with G-CSF, EPO and VEGF
95
using the protein/dextran/PLGA microsphere delivery system could promote
96
neovascularization, and was more effective than sigle factor therapy in a rat hindlimb
97
ischemia model. VEGF/dextran/PLGA, G-CSF/dextran/PLGA and EPO/dextran/PLGA
98
microspheres were prepared. The morphology and in vitro release characteristics of
99
microspheres were studies. Fibrin gel simultaneously loaded with these three types of
100
microspheres was injected into the muscle tissue of the hindlimb ischemia model in rats, and
101
neovascularization in ischemic tissue was detected.
102 103
Methods
104
Production of protein/dextran/PLGA microspheres
105
As previously described [14, 16], protein/dextran/PLGA microspheres were prepared. Human
106
G-CSF, EPO or rat VEGF 165 (PeproTech), dextran (Sigma-Aldrich), and PEG
107
(Sigma-Aldrich) were mixed at a mass ratio of 1:5:50, stirred for 1 min at 1500 rpm,
108
refrigerated for 12 h at -80
109
washed three times with dichloromethane. The final protein-dextran microparticles were
110
collected and evaporated for 24 h, then suspended in the PLGA (15%, w/w) (Sigma-Aldrich)
111
solution dissolved in dichloromethane, stirred immediately for 60 s, emulsified into NaCl
, and then lyophilized for 24 h. The lyophilized powder was
6
112
(5%, w/w) solution added with polyvinyl alcohol (1%, w/w) (Sigma-Aldrich), and stirred at
113
2000 rpm. Initial protein/dextran/PLGA microspheres were obtained. In order to solidify the
114
protein/dextran/PLGA microspheres, the above microspheres emulsion was added to NaCl
115
(10%, w/w) solution at 4
116
VEGF/dextran/PLGA, G-CSF/dextran/PLGA and EPO/dextran/PLGA microspheres were
117
washed with pure water, and then lyophilized for 24 h.
, and stirred for 4 h at 150 rpm. Finally, the hardened
118 119
Morphology of protein/dextran/PLGA microspheres
120
The morphological characteristics of G-CSF/dextran/PLGA, EPO/dextran/PLGA and
121
VEGF/dextran/PLGA microspheres were observed by scanning electron microscopy (SEM).
122
Before image scanning, gold was deposited on the surface of microspheres by vapor
123
deposition.
124 125
Encapsulation efficiency of protein/dextran/PLGA microspheres
126
G-CSF/dextran/PLGA, EPO/dextran/PLGA and VEGF/dextran/PLGA microspheres were
127
dissolved in dichloromethane respectively, and centrifuged at 10,000 rpm for 5 min. The
128
supernatant was removed. The insoluble pellets were collected and dissolved in phosphate
129
buffer saline (PBS). VEGF, G-CSF and EPO ELISA kits (R&D Systems) were used to
130
determine the concentration of released factors. The encapsulation efficiency of
131
protein/dextran/PLGA microspheres was evaluated by the following formula: encapsulation
132
efficiency =Wa/Wt×100%. In this formula, Wa refers to the actual weight of protein
133
microencapsulated into dextran/PLGA microspheres, and Wt refers to the theoretical weight
7
134
of protein microencapsulated into dextran/PLGA microspheres.
135 136
Release profile of protein/dextran/PLGA microspheres
137
The 20mg of G-CSF/dextran/PLGA, EPO/dextran/PLGA and VEGF/dextran/PLGA
138
microspheres were suspended in 2mL PBS, respectively, and continuously oscillated at 37°C.
139
The released PBS was collected and the same amount of fresh PBS was added every two days.
140
VEGF, G-CSF and EPO ELISA kits (R&D Systems) were used to determine the contents of
141
G-CSF, EPO and VEGF. For each type of microsphere, release analysis was repeated six
142
times. The cumulative release amount of proteins was plotted against time for each sampling
143
interval time to get the cumulative release profiles.
144 145
Rat ischemic hindlimb model and grouping
146
Sprague-Dawley rats (male, weight 200g) were anesthetized by intraperitoneal injection of 60
147
mg/kg ketamine. From the inguinal ligament to the knee, a vertical longitudinal incision was
148
made in the left hindlimb. The femoral artery and its branches were dissected, ligated and
149
completely resected. Fibrin gel was prepared by mixing 2 ml of the thrombin component (400
150
IU/mL), 2 ml of the sealer protein containing fibrinogen, factor XIII and aprotinin, and
151
protein/dextran/PLGA microspheres. One day after operation, 60 rats were randomly divided
152
into five groups (12 in each group): G+E+V, in which the rats were treated with intramuscular
153
injection of fibrin gel loaded with G-CSF/dextran/PLGA, EPO/dextran/PLGA and
154
VEGF/dextran/PLGA microspheres in the anterior tibialis muscle (volume = 200µl,
155
G-CSF=70 µg/rat, EPO=2000U/rat, VEGF=70 µg/rat); V, in which the rats got treatment with
8
156
fibrin gel loaded with VEGF/dextran/PLGA microspheres; G, in which the rats got treatment
157
with fibrin gel loaded with G-CSF/dextran/PLGA microspheres; E, in which the rats got
158
treatment with fibrin gel loaded with EPO/dextran/PLGA microspheres; C, as a control group,
159
in which the rats got treatment with fibrin gel loaded with empty dextran/PLGA microspheres.
160
The dose of growth factors was based on previous similar studies [4, 5, 8, 17-21]. The
161
protocols for all experiments involving animal surgery were approved by the institutional
162
animal care and use committee of Nanchang University.
163 164
Western blot, histological and immunohistochemical analysis
165
On day 7 and 28 post-treatment, tibialis anterior muscle specimens were harvested and split
166
into two parts for use in both Western blot and histological analysis. Muscle tissue was cut
167
into small pieces, grinded using a homogenizer with lysis buffer containing 1 mmol/L DTT,
168
400 µmol/L PMSF, 20 mmol/L Hepes (pH 7.4), 1 mmol/L Na3VO4, 2 mmol/L EDTA, 10%
169
glycerol, 1% Triton X-100, 2 µmol/L leupeptin and 10 units/mL aprotinin (Sigma), and
170
incubated at 4°C for 30 min. Lysates were centrifuged at 10,000 rpm at 4°C for 10 min and
171
the supernatant was collected. Protein concentration was determined by BCA method. The 20
172
ug protein extract from each sample was heated at 95°C for 10 min in a loading buffer,
173
separated onto 10% SDS-PAGE, transferred to 0.22 µm PVDF membranes (Millipore),
174
blocked in 5% non-fat milk for 2 hours at room temperature, and incubated overnight at 4 °C
175
with the respective primary antibodies: rabbit antibodies against stromal cell derived factor 1
176
(SDF-1) (Abcam), B-cell lymphoma-2 (Bcl-2) (Abcam), β-actin (Abcam), Bcl-2 associated x
177
protein (Bax) (Abcam) and C-X-C chemokine receptor 4 (CXCR4) (Abcam). After washing
9
178
three times in TBST, the membranes were incubated at room temperature with a
179
HRP-conjugated goat anti-rabbit secondary antibody (Vector Laboratories) for 1 h followed
180
by washing three times in TBST, and the protein bands were visualized using a commercial
181
ECL kit (Amersham) and Kodak film. The protein bands of SDF-1, Bcl-2, Bax, CXCR4 and
182
β-actin were quantitatively analyzed using ImageJ Software.
183
Muscle tissue was embedded in OCT compound and snap-frozen. They were cut into
184
serial sections 6 µm thick. In order to detect capillary ECs, the sections were stained with
185
alkaline phosphatase. In order to detect smooth muscle α-actin (SMA) positive vessels,
186
immunohistochemical staining was performed with a rabbit antibody against SMA (Abcam).
187
The stained capillaries and SMA+ vessels on sections were quantitatively analyzed using
188
ImageJ Software. And immunohistochemical staining with primary antibodies against SDF-1,
189
Bcl-2, CXCR4 and proliferating cell nuclear antigen (PCNA) (Abcam) were performed on
190
sections. Non-immune IgG of the corresponding species was used as negative control to
191
confirm the specificity of primary antibody. The sections were stained according to the
192
manufacturer’s protocols.
193 194
Statistics
195
SPSS 13.0 (SPSS Inc, Chicago) was used to analyze the data which were presented as
196
means±S.E. Comparison of means was achieved using Student’s t-test and one-way ANOVA
197
with Tukey’s honestly significant differences test. The significance was assigned at P <0.05.
198 199
Results
200
Morphological characteristics of protein/dextran/PLGA microspheres
10
201
The morphological characteristics of the protein/dextran/PLGA microspheres loaded with
202
G-CSF, EPO or VEGF were evaluated with SEM scanning method. Protein/dextran/PLGA
203
microspheres were spherical with smooth surface and diameters of 40-120 µm (Figure 1).
204 205
Encapsulation efficiency of protein/dextran/PLGA microspheres
206
ELISA kits were used to evaluate the protein encapsulation efficiency of
207
protein/dextran/PLGA microspheres. The protein encapsulation efficiencies of
208
G-CSF/dextran/PLGA, EPO/dextran/PLGA and VEGF/dextran/PLGA microspheres were
209
84%±4.2, 85%±4.0, 82%±3.8 (w/w), respectively.
210 211
Release profile of protein/dextran/PLGA microspheres
212
Protein/dextran/PLGA microspheres released angiogenic factors for more than 4 weeks (Fig.
213
1). The cumulative releases of G-CSF, EPO and VEGF were approximately 89.5%, 90.3%
214
and 91.2% respectively, and the amounts of burst release were about 23.5%, 25.8% and
215
26.5% respectively during the first two days.
216 217
Neovascularization in ischemic muscle
218
To detect capillary ECs, alkaline phosphatase staining was performed on ischemic muscle
219
sections. As shown in Fig. 2 and Fig. 3, the capillary density (number/muscle fiber) was
220
significantly higher in G, E, V and G+E+V groups than in control group (p
221
capillary density in G+E+V group was the highest.
222
0.05), and the
At post-treatment day 28, to detect SMA+ vessels, immunohistochemical analysis with
11
223
an antibody against SMA were performed on ischemic muscle sections. As showed Fig. 2 and
224
Fig. 3, the density of SMA positive vessels in G, V and G+E+V groups was significantly
225
higher than that in the control group (p
226
group was the highest.
0.05), and the density of SMA+ vessels in G+E+V
227 228
Enhanced Bcl-2 expression and cellular proliferation
229
To detect the expression of Bcl-2, at post-treatment day 7, immunohistochemical analysis and
230
Western blot analysis were performed using antibodies against Bcl-2 and Bax. As shown in
231
Fig. 4 and Fig. 5, it seemed that G-CSF, EPO and VEGF all could promote the expression of
232
Bcl-2. The Bcl-2 expression in group G+E+V was significantly higher than that in other
233
groups (p
0.05).
234
To examine cellular proliferative activity, immunohistochemical analysis with an
235
antibody against PCNA was performed. In Fig. 4, it seemed that G-CSF, EPO and VEGF all
236
seemed to promote cellular proliferation in varying degrees. The cellular proliferative activity
237
in G+E+V group was higher than that in other groups.
238 239
Enhanced SDF-1 expression and CXCR4+ cells incorporation
240
To examine the SDF-1 expression, at post-treatment day 7, immunohistochemical analysis
241
and Western blot analysis with an antibody against SDF-1 were performed. As shown in Fig.
242
6 and Fig. 7, it seemed that EPO alone promoted the expression of SDF-1 in group E. The
243
expression of SDF-1 in group G+E+V was significantly higher than that in other groups (p
244
0.05).
12
245
In Fig. 2, muscle tissues with HE staining showed varying degrees of cellular infiltration
246
in each group. In order to detect the incorporation of CXCR4+ cells in ischemic tissues,
247
immunohistochemical analysis and Western blot analysis using an antibody against CXCR4
248
were performed. As shown in Fig. 6 and Fig. 7, it was found that combination therapy with
249
G-CSF, EPO and VEGF could promote the incorporation of CXCR4+ cells more effectively
250
than single factor therapy.
251 252
Discussion
253
An important obstacle to growth factor-based therapeutic neovascularization is the short
254
half-life of pro-angiogenic growth factors. To solve this problem, a variety of sustained drug
255
release systems have been developed to control the release of pro-angiogenic factors and
256
protect them from degradation. PLGA microspheres are the most widely used [17, 22], and
257
firstly approved by the Food and Drug Administration of the United States. However, during
258
the microencapsulation process of PLGA microspheres, growth factors tend to denaturate or
259
aggregate due to the effect of water/oil or water/air interfacial tension [9, 10]. In order to
260
avoid these defects, an effective method is to pre-load proteins into dextran microparticles,
261
and then microencapsulate these protein/dextran microparticles into PLGA microspheres by a
262
solid-in-oil-in-water method [10, 13-15]. It was found that these protein/dextran/PLGA
263
microspheres produced by solid-in-oil-in-water method had higher encapsulation efficiency,
264
lower initial burst release, longer release period and better bioactivity preservation than
265
protein-PLGA microspheres produced by water-in-oil-in-water method. In this study, on the
266
basis of the combination of PLGA microspheres and protein/dextran microparticles,
267
VEGF/dextran/PLGA, G-CSF/dextran/PLGA and EPO/dextran/PLGA microspheres were
13
268
produced. The results showed that more than 80% of drug-loaded growth factors could be
269
released from microspheres efficiently over 4 weeks in vitro. In the rat hindlimb ischemia
270
model, it was also found that the released VEGF, G-CSF and EPO all could significantly
271
promote neovascularization by increasing the density of capillaries or SMA+ vessels. As
272
shown in our results, protein/dextran/PLGA microspheres sustained release system was an
273
ideal drug delivery system for combinational therapeutic neovascularization.
274
In this study, it was found that VEGF, G-CSF and EPO all significantly increased the
275
expression of Bcl-2, and the number of Bcl-2+/PCNA+ proliferating cells in the ischemic
276
muscle. First of all, VEGF, as a mitogen to ECs, can inhibit the apoptosis of ECs and
277
stimulate the proliferation of ECs [17, 19, 23]. VEGF can promote the expression of Bcl-2 in
278
ECs and increase the density of PCNA+ ECs. Secondly, G-CSF can facilitate mobilization,
279
recruitment and survival of bone marrow and peripheral bold derived leukocytes, which can
280
express pro-angiogenic factors such as VEGF and PDGF to promote neovascularization [6,
281
18, 24]. Therefore, G-CSF can also promote the expression of Bcl-2 and PCNA in leukocytes
282
and other effector cells [21]. Thirdly, EPO is a hematopoietic cytokine, which can promote
283
the proliferation, differentiation and mobilization of erythroid progenitor cells. EPO receptor
284
(EPOR) is expressed not only in erythroid progenitor cells, but also in all sorts of
285
non-hematopoietic cells, such as ECs [7, 20]. EPO/EPOR can activate the signal transduction
286
proteins such as Janus kinase 2, signal transducer and activator of transcription which can
287
control cellular proliferation, survival and gene expression [7, 20]. EPO/EPOR can enhance
288
the proliferation, survival and migration of ECs, protect them from ischemia and apoptosis,
289
and promote angiogenesis in vitro and in vivo [4, 7, 20, 25]. Similarly, EPO could also
14
290
enhance the expression of Bcl-2 and PNCA in ECs. G-CSF, VEGF and EPO all could
291
increase the number of Bcl-2+/PCNA+ expression cells in ischemic muscle, thus protecting
292
cells from apoptosis, promoting cell proliferation, and inducing angiogenesis. Compared with
293
monotherapy with G-CSF, EPO or VEGF alone, combined treatment with G-CSF, EPO and
294
VEGF could significantly promote the expression of Bcl-2 and PCNA.
295
In this study, it was found that EPO/dextran/PLGA microspheres could significantly
296
promote SDF-1 expression and increase the incorporation of CXCR4+ cells in ischemic
297
tissues. Lin et al. [26] also found that the increased SDF-1 expression was mediated by EPO,
298
since treatment with an antibody against EPOR markedly blocked this effect. It seemed that
299
EPO directly affected the expression of SDF-1. Other studies found that EPO induced the
300
release of SDF-1 from hematopoietic cells, particularly platelets, and contributed to
301
neovascularization of ischemic limb [5, 27, 28]. CXCR4, as a main SDF-1 receptor, is widely
302
and constitutively expressed by bone marrow derived cells [28]. CXCR4 positive
303
pro-angiogenic cells include hematopoietic cells, endothelial progenitor cells, smooth muscle
304
progenitor cells and pericytes. They all have the ability to promote neovascularization
305
directly or indirectly [29]. SDF-1/CXCR4 can regulate some key aspects of blood vessel
306
development, stem cells recruitment and postnatal vasculogenesis [29-32]. It was concluded
307
that EPO could play an avtive role in neovascularization by increasing the expression of
308
SDF-1.
309
VEGF receptor 2 (VEGFR2) and CXCR4 positive cells represent specific subgrouops of
310
pro-angiogenic cells, have overlapping phenotypes, and are mobilized by VEGF-A and
311
SDF-1, respectively [28]. VEGF-A can promote angiogenesis and bone marrow cell
15
312
recruitment by activating SDF-1–CXCR4 pathway [19, 28]. EPO/EPOR plays an important
313
role in angiogenesis induced by ischemia through the upregulation of VEGF/VEGFR system
314
which promotes the recruitment of endothelial progenitor cells and other bone marrow
315
derived proangiogenic cells, and enhances neovascularization in ischemic tissue [6, 28]. In
316
summary, EPO and VEGF seem to have synergistic effects in mobilizing and recruiting
317
CXCR4+ cells to promote neovascularization in ischemic tissues [28].
318
G-CSF is a hematopoietic growth factor that promotes mobilization and proliferation of
319
bone marrow derived hematopoietic stem cells and progenitor cells. By mobilizing and
320
stimulating bone marrow-derived monocytes and progenitor cells to secrete various
321
angiogenic factors or differentiate into endothelial cells, smooth muscle cells or pericytes,
322
G-CSF can also promote the angiogenesis, arteriogenesis and vasculogenesis [6, 18, 24].
323
Through the CXCR4/SDF-1 axis, G-CSF could mobilize and recruitt CXCR4+ cells into
324
ischemic tissue to promote neovascularization in ischemic tissues.
325
The above mechanisms are summarized as follows. Firstly, G-CSF can mobilize
326
hematopoietic cells and progenitor cells, including CXCR4+ cells from bone marrow.
327
Secondly, as a source of SDF-1, these mobilized hematopoietic cells can increase the
328
expression of SDF-1 activated by EPO. More SDF-1 expression can recruit more CXCR4+
329
cells. Thirdly, because of the overlapping phenotypes of VEGFR2+ and CXCR4+ in some
330
cells, VEGF can also recruit CXCR4+ cells. Taking these effects together, G-CSF, EPO and
331
VEGF have synergistic effects on the SDF-1 expression, mobilization and recruitment of
332
CXCR4 positive cells which are beneficial to neovascularization. In this study, it was found
333
that combined treatment with G-CSF, EPO and VEGF had stronger effects on the expression
16
334
of SDF-1, and incorporation of CXCR4+ cells in ischemic tissues than monotherapy with
335
VEGF, EPO or G-CSF alone. Combined therapy could achieve better neovascularization
336
effect than monotherapy.
337
One limitation of this study was the deficiency of more in-depth mechanistic study and
338
discussion. Another limitation was the lack of evaluation of systemic toxicity and side effects.
339
Still another limitation was that this study utilized a small animal model. Further studies were
340
needed to repeat these experiments in large animal models, and assess the clinical potential of
341
this treatment.
342 343
Conclusion
344
The release of G-CSF, EPO and VEGF from protein/dextran/PLGA microspheres lasted for
345
more than 4 weeks in vitro. Combined therapy with VEGF, EPO and G-CSF using
346
dextran/PLGA microspheres had synergistic effects in promoting the expression of Bcl-2 and
347
cellular proliferation, increasing SDF-1 expression and CXCR4+ cells incorporation in
348
ischemic muscle. Compared with monotherapy with VEGF, EPO or G-CSF alone, this
349
combined therapy had more significant neovascularization effect. It may be a promising
350
treatment for ischemic vascular diseases such as cardiac ischemia, limb ischemia, and wound
351
healing.
352 353
Source of Funding
354
This work was supported by grants from the National Natural Science Foundation of China
355
(81460083, 81860094), Natural Science Foundation of Jiangxi Province (20142BAB215034,
17
356
20141BBG70032, 20152ACB21026).
357 358
Compliance with Ethical Standards
359
Conflicts of Interest
360
conflict of interest in relation to this article.
361
Ethical Approval
362
the care and use of animals were followed.
All authors have approved the final article and declare no potential
All applicable international, national, and/or institutional guidelines for
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Figure legends
475
Figure 1 A, A scanning electron micrograph of protein/dextran/PLGA microspheres. B, In
476
vitro release profiles of pro-angiogenic growth factors from protein/dextran/PLGA
477
microspheres.
478
Figure 2 Representative microscopic photographs of alkaline phosphatase staining to detect
479
capillary (AP), immunostain with an antibody against smooth muscle α-actin to detect SMA+
480
vessels (α-SMA), and staining with hematoxylin and eosin (HE). C group received empty
481
dextran/PLGA microspheres as control group; G group received G-CSF/dextran/PLGA
482
microspheres; E group received EPO/dextran/PLGA microspheres; V group received
483
VEGF/dextran/PLGA microspheres; G+E+V group three types of microspheres.
484
Figure 3 A, Densities of capillary in ischemic muscle obtained from the rats at post-treatment
485
day 7 and day 28. B, Densities of α-SMA+ vessel at post-treatment day 28. C group received
486
empty dextran/PLGA microspheres as control group; G group received G-CSF/dextran/PLGA
487
microspheres; E group received EPO/dextran/PLGA microspheres; V group received
488
VEGF/dextran/PLGA microspheres; G+E+V group three types of microspheres.
489
Quantification of capillary density as number of capillaries per fiber on the tissue sections.
21
490
Quantification of α-SMA positive vessels density as number of SMA+ vessels per fiber. *
491
p<0.05 vs. group C, # p<0.05 vs. group G+E+V.
492
Figure 4 Representative microscopic photographs of immunostaining with antibodies against
493
Bcl-2 and PCNA. C group received empty dextran/PLGA microspheres as control group; G
494
group received G-CSF/dextran/PLGA microspheres; E group received EPO/dextran/PLGA
495
microspheres; V group received VEGF/dextran/PLGA microspheres; G+E+V group three
496
types of microspheres.
497
Figure 5 Western blotting showing the expression of Bcl-2 and PCNA. Quantification of
498
protein expression in bar graphs. Expression was normalized to loading control GAPDH, and
499
expressed relative to its corresponding GAPDH. C group received empty dextran/PLGA
500
microspheres as control group; G group received G-CSF/dextran/PLGA microspheres; E
501
group received EPO/dextran/PLGA microspheres; V group received VEGF/dextran/PLGA
502
microspheres; G+E+V group three types of microspheres. * p<0.05 vs. group C, # p<0.05 vs.
503
group G+E+V.
504
Figure 6 Representative microscopic photographs of immunostaining with antibodies against
505
SDF-1 and CXCR4. C group received empty dextran/PLGA microspheres as control group;
506
G group received G-CSF/dextran/PLGA microspheres; E group received EPO/dextran/PLGA
507
microspheres; V group received VEGF/dextran/PLGA microspheres; G+E+V group three
508
types of microspheres.
509
Figure 7 Western blotting showing the expression of SDF-1 and CXCR4. Quantification of
510
protein expression in bar graphs. Expression was normalized to loading control GAPDH, and
511
expressed relative to its corresponding GAPDH. C group received empty dextran/PLGA
22
512
microspheres as control group; G group received G-CSF/dextran/PLGA microspheres; E
513
group received EPO/dextran/PLGA microspheres; V group received VEGF/dextran/PLGA
514
microspheres; G+E+V group three types of microspheres. * p<0.05 vs. group C, # p<0.05 vs.
515
group G+E+V.