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• W2891526049 abstract "The application of endothelial progenitor cells (EPCs) for the revascularization of ischemic tissues, such as after myocardial infarction, stroke, and acute limb ischemia, has a huge clinical potential. However, the low retention and engraftment of EPCs as well as the poor survival of migrated stem cells in ischemic tissues still hamper the successful clinical application. Thus, in this study, we engineered, for the first time, murine EPCs with synthetic mRNAs to transiently produce proangiogenic factors vascular endothelial growth factor-A (VEGF-A), stromal cell-derived factor-1α (SDF-1α), and angiopoietin-1 (ANG-1). After the transfection of cells with synthetic mRNAs, significantly increased VEGF-A, SDF-1α, and ANG-1 protein levels were detected compared to untreated EPCs. Thereby, mRNA-engineered EPCs showed significantly increased chemotactic activity versus untreated EPCs and resulted in significantly improved attraction of EPCs. Furthermore, ANG-1 mRNA-transfected EPCs displayed a strong wound-healing capacity. Already after 12 hr, 94% of the created wound area in the scratch assay was closed compared to approximately 45% by untreated EPCs. Moreover, the transfection of EPCs with ANG-1 or SDF-1α mRNA also significantly improved the in vitro tube formation capacity; however, the strongest effect could be detected with EPCs simultaneously transfected with VEGF-A, SDF-1α, and ANG-1 mRNA. In the in vivo chicken chorioallantoic membrane (CAM) assay, EPCs transfected with ANG-1 mRNA revealed the strongest angiogenetic potential with significantly elevated vessel density and total vessel network length. In conclusion, this study demonstrated that EPCs can be successfully engineered with synthetic mRNAs encoding proangiogenic factors to improve their therapeutic angiogenetic potential in patients experiencing chronic or acute ischemic disease. The application of endothelial progenitor cells (EPCs) for the revascularization of ischemic tissues, such as after myocardial infarction, stroke, and acute limb ischemia, has a huge clinical potential. However, the low retention and engraftment of EPCs as well as the poor survival of migrated stem cells in ischemic tissues still hamper the successful clinical application. Thus, in this study, we engineered, for the first time, murine EPCs with synthetic mRNAs to transiently produce proangiogenic factors vascular endothelial growth factor-A (VEGF-A), stromal cell-derived factor-1α (SDF-1α), and angiopoietin-1 (ANG-1). After the transfection of cells with synthetic mRNAs, significantly increased VEGF-A, SDF-1α, and ANG-1 protein levels were detected compared to untreated EPCs. Thereby, mRNA-engineered EPCs showed significantly increased chemotactic activity versus untreated EPCs and resulted in significantly improved attraction of EPCs. Furthermore, ANG-1 mRNA-transfected EPCs displayed a strong wound-healing capacity. Already after 12 hr, 94% of the created wound area in the scratch assay was closed compared to approximately 45% by untreated EPCs. Moreover, the transfection of EPCs with ANG-1 or SDF-1α mRNA also significantly improved the in vitro tube formation capacity; however, the strongest effect could be detected with EPCs simultaneously transfected with VEGF-A, SDF-1α, and ANG-1 mRNA. In the in vivo chicken chorioallantoic membrane (CAM) assay, EPCs transfected with ANG-1 mRNA revealed the strongest angiogenetic potential with significantly elevated vessel density and total vessel network length. In conclusion, this study demonstrated that EPCs can be successfully engineered with synthetic mRNAs encoding proangiogenic factors to improve their therapeutic angiogenetic potential in patients experiencing chronic or acute ischemic disease. The insufficient perfusion of tissues or organs with blood results in ischemia and often leads to conditions such as stroke, myocardial infarction, or acute limb ischemia. During ischemia, the affected tissues are damaged due to hypoxia and the lack of nutrient supply and waste removal. Thus, the formation of new blood vessels by angiogenesis and vasculogenesis is required. During angiogenesis, new blood vessels are formed from pre-existing vessels and, during vasculogenesis, de novo blood vessel formation occurs through bone marrow-derived endothelial progenitor cells (EPCs).1Takahashi T. Kalka C. Masuda H. Chen D. Silver M. Kearney M. Magner M. Isner J.M. Asahara T. Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization.Nat. Med. 1999; 5: 434-438Crossref PubMed Scopus (46) Google Scholar Thereby, the blood supply can be restored to ischemic tissues, the tissue damage can be reduced, and, as a result, the function of affected tissues or organs can be improved. EPCs are circulating stem cells in the bloodstream, and they are recruited by chemokines, which are secreted by injured and activated cells at the injury site to induce revascularization.2Asahara T. Masuda H. Takahashi T. Kalka C. Pastore C. Silver M. Kearne M. Magner M. Isner J.M. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization.Circ. Res. 1999; 85: 221-228Crossref PubMed Scopus (2924) Google Scholar EPCs settle and differentiate at sites of vascular lesions, and they appear to participate in vascular repair and homeostasis.3Asahara T. Kawamoto A. Masuda H. Concise review: Circulating endothelial progenitor cells for vascular medicine.Stem Cells. 2011; 29: 1650-1655Crossref PubMed Scopus (327) Google Scholar Previous studies even identified EPCs as biomarkers in cardiovascular diseases, and a decline of EPC numbers and dysfunction were related to ischemic diseases.4Sen S. McDonald S.P. Coates P.T. Bonder C.S. Endothelial progenitor cells: novel biomarker and promising cell therapy for cardiovascular disease.Clin. Sci. (Lond.). 2011; 120: 263-283Crossref PubMed Scopus (177) Google Scholar, 5Kachamakova-Trojanowska N. Bukowska-Strakova K. Zukowska M. Dulak J. Jozkowicz A. The real face of endothelial progenitor cells - Circulating angiogenic cells as endothelial prognostic marker?.Pharmacol. Rep. 2015; 67: 793-802Crossref PubMed Scopus (22) Google Scholar Several preclinical studies have demonstrated the vasculogenic and angiogenic potential,6Kawamoto A. Gwon H.C. Iwaguro H. Yamaguchi J.I. Uchida S. Masuda H. Silver M. Ma H. Kearney M. Isner J.M. Asahara T. Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia.Circulation. 2001; 103: 634-637Crossref PubMed Scopus (1119) Google Scholar, 7Minami Y. Nakajima T. Ikutomi M. Morita T. Komuro I. Sata M. Sahara M. Angiogenic potential of early and late outgrowth endothelial progenitor cells is dependent on the time of emergence.Int. J. Cardiol. 2015; 186: 305-314Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar as well as beneficial paracrine effects, of transplanted EPCs in the treatment of ischemic diseases.8Fazel S. Cimini M. Chen L. Li S. Angoulvant D. Fedak P. Verma S. Weisel R.D. Keating A. Li R.K. Cardioprotective c-kit+ cells are from the bone marrow and regulate the myocardial balance of angiogenic cytokines.J. Clin. Invest. 2006; 116: 1865-1877Crossref PubMed Scopus (461) Google Scholar, 9Chong M.S.K. Ng W.K. Chan J.K.Y. Concise Review: Endothelial Progenitor Cells in Regenerative Medicine: Applications and Challenges.Stem Cells Transl. Med. 2016; 5: 530-538Crossref PubMed Scopus (134) Google Scholar However, the success of clinical applications is limited due to low retention and engraftment of EPCs as well as the poor survival of migrated EPCs in ischemic tissues.10Terrovitis J.V. Smith R.R. Marbán E. Assessment and optimization of cell engraftment after transplantation into the heart.Circ. Res. 2010; 106: 479-494Crossref PubMed Scopus (252) Google Scholar, 11Lipiec P. Krzemińska-Pakuła M. Plewka M. Kuśmierek J. Płachcińska A. Szumiński R. Robak T. Korycka A. Kasprzak J.D. Impact of intracoronary injection of mononuclear bone marrow cells in acute myocardial infarction on left ventricular perfusion and function: a 6-month follow-up gated 99mTc-MIBI single-photon emission computed tomography study.Eur. J. Nucl. Med. Mol. Imaging. 2009; 36: 587-593Crossref PubMed Scopus (26) Google Scholar Furthermore, the low quantity and quality of isolated autologous EPCs is challenging for clinical applications.12Sukmawati D. Tanaka R. Introduction to next generation of endothelial progenitor cell therapy: a promise in vascular medicine.Am. J. Transl. Res. 2015; 7: 411-421PubMed Google Scholar Thus, multiple animal and clinical studies with EPCs demonstrated different results in their effectiveness to treat ischemic diseases.9Chong M.S.K. Ng W.K. Chan J.K.Y. Concise Review: Endothelial Progenitor Cells in Regenerative Medicine: Applications and Challenges.Stem Cells Transl. Med. 2016; 5: 530-538Crossref PubMed Scopus (134) Google Scholar, 13Zhang H. van Olden C. Sweeney D. Martin-Rendon E. Blood vessel repair and regeneration in the ischaemic heart.Open Heart. 2014; 1: e000016Crossref PubMed Scopus (29) Google Scholar Consequently, novel strategies are needed to enhance the number and function of EPCs and to obtain a successful autologous EPC therapy for a more efficient tissue regeneration. In previous studies, various growth factors, such as vascular endothelial growth factor (VEGF)14Takeshita S. Zheng L.P. Brogi E. Kearney M. Pu L.Q. Bunting S. Ferrara N. Symes J.F. Isner J.M. Therapeutic angiogenesis. A single intraarterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hind limb model.J. Clin. Invest. 1994; 93: 662-670Crossref PubMed Scopus (1000) Google Scholar or fibroblast growth factor (FGF),15Baffour R. Berman J. Garb J.L. Rhee S.W. Kaufman J. Friedmann P. Enhanced angiogenesis and growth of collaterals by in vivo administration of recombinant basic fibroblast growth factor in a rabbit model of acute lower limb ischemia: dose-response effect of basic fibroblast growth factor.J. Vasc. Surg. 1992; 16: 181-191Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar were applied to improve the revascularization of tissues.16Sun X.-T. Ding Y.-T. Yan X.-G. Wu L.-Y. Li Q. Cheng N. Qiu Y.D. Zhang M.Y. Angiogenic synergistic effect of basic fibroblast growth factor and vascular endothelial growth factor in an in vitro quantitative microcarrier-based three-dimensional fibrin angiogenesis system.World J. Gastroenterol. 2004; 10: 2524-2528Crossref PubMed Scopus (46) Google Scholar Local levels of proangiogenic proteins were upregulated by delivering recombinant proteins17Wang J.H. Xu Y.M. Fu Q. Song L.J. Li C. Zhang Q. Xie M.K. Continued sustained release of VEGF by PLGA nanospheres modified BAMG stent for the anterior urethral reconstruction of rabbit.Asian Pac. J. Trop. Med. 2013; 6: 481-484Crossref PubMed Scopus (20) Google Scholar, 18Chappell J.C. Song J. Burke C.W. Klibanov A.L. Price R.J. Targeted delivery of nanoparticles bearing fibroblast growth factor-2 by ultrasonic microbubble destruction for therapeutic arteriogenesis.Small. 2008; 4: 1769-1777Crossref PubMed Scopus (76) Google Scholar or genes19Baumgartner I. Pieczek A. Manor O. Blair R. Kearney M. Walsh K. Isner J.M. Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia.Circulation. 1998; 97: 1114-1123Crossref PubMed Scopus (1072) Google Scholar, 20Kuliszewski M.A. Kobulnik J. Lindner J.R. Stewart D.J. Leong-Poi H. Vascular gene transfer of SDF-1 promotes endothelial progenitor cell engraftment and enhances angiogenesis in ischemic muscle.Mol. Ther. 2011; 19: 895-902Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar using nano- or microparticles,17Wang J.H. Xu Y.M. Fu Q. Song L.J. Li C. Zhang Q. Xie M.K. Continued sustained release of VEGF by PLGA nanospheres modified BAMG stent for the anterior urethral reconstruction of rabbit.Asian Pac. J. Trop. Med. 2013; 6: 481-484Crossref PubMed Scopus (20) Google Scholar, 18Chappell J.C. Song J. Burke C.W. Klibanov A.L. Price R.J. Targeted delivery of nanoparticles bearing fibroblast growth factor-2 by ultrasonic microbubble destruction for therapeutic arteriogenesis.Small. 2008; 4: 1769-1777Crossref PubMed Scopus (76) Google Scholar direct injection into the target tissue,19Baumgartner I. Pieczek A. Manor O. Blair R. Kearney M. Walsh K. Isner J.M. Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia.Circulation. 1998; 97: 1114-1123Crossref PubMed Scopus (1072) Google Scholar ultrasound-targeted microbubble destruction (UTMD),20Kuliszewski M.A. Kobulnik J. Lindner J.R. Stewart D.J. Leong-Poi H. Vascular gene transfer of SDF-1 promotes endothelial progenitor cell engraftment and enhances angiogenesis in ischemic muscle.Mol. Ther. 2011; 19: 895-902Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 21Leong-Poi H. Kuliszewski M.A. Lekas M. Sibbald M. Teichert-Kuliszewska K. Klibanov A.L. Stewart D.J. Lindner J.R. Therapeutic arteriogenesis by ultrasound-mediated VEGF165 plasmid gene delivery to chronically ischemic skeletal muscle.Circ. Res. 2007; 101: 295-303Crossref PubMed Scopus (151) Google Scholar or sustained release from implants.22Swanson N. Hogrefe K. Javed Q. Malik N. Gershlick A.H. Vascular endothelial growth factor (VEGF)-eluting stents: in vivo effects on thrombosis, endothelialization and intimal hyperplasia.J. Invasive Cardiol. 2003; 15: 688-692PubMed Google Scholar The use of recombinant proteins is costly and it is difficult to maintain adequate protein levels in the ischemic regions due to their relatively short half-lives.23Gupta R. Tongers J. Losordo D.W. Human studies of angiogenic gene therapy.Circ. Res. 2009; 105: 724-736Crossref PubMed Scopus (237) Google Scholar Therefore, gene therapy with viral and non-viral vectors was used as an alternative strategy to express the desired proangiogenic proteins, and it has shown to be promising, for example, for the treatment of myocardial ischemia.13Zhang H. van Olden C. Sweeney D. Martin-Rendon E. Blood vessel repair and regeneration in the ischaemic heart.Open Heart. 2014; 1: e000016Crossref PubMed Scopus (29) Google Scholar, 23Gupta R. Tongers J. Losordo D.W. Human studies of angiogenic gene therapy.Circ. Res. 2009; 105: 724-736Crossref PubMed Scopus (237) Google Scholar In recent years, the expression of exogenous proteins by the delivery of synthetic mRNAs has gained great importance as an alternative strategy to the viral vector or plasmid-based gene delivery methods.24Sahin U. Karikó K. Türeci Ö. mRNA-based therapeutics--developing a new class of drugs.Nat. Rev. Drug Discov. 2014; 13: 759-780Crossref PubMed Scopus (1061) Google Scholar In contrast to genome-integrating gene delivery methods, the application of synthetic mRNA is a non-integrating method with no risk of insertional mutagenesis. Since the mRNA does not need to enter the nucleus, the synthetic mRNA can be efficiently delivered in dividing as well as non-dividing cells. The transfection of synthetic mRNA leads to the transient production of exogenous proteins in the cells, and, after the natural degradation of mRNA, no footprint is left in the cells. The most intensively studied growth factor for the induction of vascularization and angiogenesis is the vascular endothelial growth factor-A (VEGF-A). It is involved in the chemotaxis, migration, and differentiation of progenitor cells; endothelial cell (EC) survival and proliferation; as well as the sprouting of vessels and vessel permeability.25Adams R.H. Alitalo K. Molecular regulation of angiogenesis and lymphangiogenesis.Nat. Rev. Mol. Cell Biol. 2007; 8: 464-478Crossref PubMed Scopus (1468) Google Scholar Among alternative splice variants of VEGF-A, VEGF-A165 is the quantitatively and qualitatively most important variant for angiogenesis.26Shibuya M. Vascular Endothelial Growth Factor (VEGF) and Its Receptor (VEGFR) Signaling in Angiogenesis: A Crucial Target for Anti- and Pro-Angiogenic Therapies.Genes Cancer. 2011; 2: 1097-1105Crossref PubMed Scopus (825) Google Scholar VEGF-A binds and activates VEGF receptors 1 and 2 (VEGFR-1 and VEGFR-2) expressed on vascular ECs and EPCs. VEGFR-2 has approximately 10-fold higher kinase activity than VEGFR-1, and the major proangiogenic signal is generated from the ligand-activated VEGFR-2.26Shibuya M. Vascular Endothelial Growth Factor (VEGF) and Its Receptor (VEGFR) Signaling in Angiogenesis: A Crucial Target for Anti- and Pro-Angiogenic Therapies.Genes Cancer. 2011; 2: 1097-1105Crossref PubMed Scopus (825) Google Scholar Angiopoietin-1 (ANG-1) is produced by peri-ECs and platelets.27Milam K.E. Parikh S.M. The angiopoietin-Tie2 signaling axis in the vascular leakage of systemic inflammation.Tissue Barriers. 2015; 3: e957508Crossref PubMed Scopus (61) Google Scholar It binds to Tie2 receptors on ECs and maintains endothelial integrity and reduces the effects of inflammation,28Brouwers J. Noviyanti R. Fijnheer R. de Groot P.G. Trianty L. Mudaliana S. Roest M. Syafruddin D. van der Ven A. de Mast Q. Platelet activation determines angiopoietin-1 and VEGF levels in malaria: implications for their use as biomarkers.PLoS ONE. 2013; 8: e64850Crossref PubMed Scopus (28) Google Scholar which prevents vascular leakage and stabilizes vessels. ANG-1 is further involved in EC migration and the reorganization of ECs.29Brindle N.P.J. Saharinen P. Alitalo K. Signaling and functions of angiopoietin-1 in vascular protection.Circ. Res. 2006; 98: 1014-1023Crossref PubMed Scopus (333) Google Scholar Stromal cell-derived factor-1α (SDF-1α, also known as CXCL12) is a chemokine that mediates the mobilization and recruitment of bone marrow-derived progenitor cells that express CXCR4 receptor on the cell surface, such as EPCs. Additionally, SDF-1α attenuates EC apoptosis and stimulates new vessel capillary tube formation, and the expression of SDF-1α is regulated by hypoxia.25Adams R.H. Alitalo K. Molecular regulation of angiogenesis and lymphangiogenesis.Nat. Rev. Mol. Cell Biol. 2007; 8: 464-478Crossref PubMed Scopus (1468) Google Scholar, 30Ho T.K. Tsui J. Xu S. Leoni P. Abraham D.J. Baker D.M. Angiogenic effects of stromal cell-derived factor-1 (SDF-1/CXCL12) variants in vitro and the in vivo expressions of CXCL12 variants and CXCR4 in human critical leg ischemia.J. Vasc. Surg. 2010; 51: 689-699Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 31Ceradini D.J. Kulkarni A.R. Callaghan M.J. Tepper O.M. Bastidas N. Kleinman M.E. Capla J.M. Galiano R.D. Levine J.P. Gurtner G.C. Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1.Nat. Med. 2004; 10: 858-864Crossref PubMed Scopus (2210) Google Scholar In this study, for the first time, murine EPCs were engineered by the exogenous delivery of synthetic mRNAs to increasingly express the proangiogenic proteins VEGF-A, SDF-1α, and ANG-1. The ability of EPCs to produce the desired proteins was analyzed after the transfection with synthetic mRNAs using ELISA. Next, the migration behavior of mRNA-engineered EPCs was investigated by chemotactic and wound-healing migration assay and compared with non-engineered EPCs in vitro. Then, the in vitro and in vivo angiogenic potential of mRNA-engineered EPCs was analyzed using tube formation and chick chorioallantoic membrane (CAM) assay. The specific length and purity of the generated PCR products and the in vitro-transcribed modified mRNAs were analyzed by agarose gel electrophoresis. After the gel electrophoresis, single bands without misamplifications and expected lengths were detected for the PCR products ANG-1 (1,794 bp), SDF-1α (567 bp), and VEGF-A (873 bp) and the resulting in vitro-transcribed mRNA containing the coding sequence, 3′ and 5′ UTR regions, and PolyA120 tail (Figure 1). After the successful production of synthetic mRNAs, the expression of specific proteins was determined. Therefore, single-mRNA transfections with 1.6 μg ANG-1, 0.8 μg VEGF-A, or 0.5 μg SDF-1α or transfections with an mRNA cocktail consisting of 1.6 μg ANG-1, 0.8 μg VEGF-A, and 0.5 μg SDF-1α were performed. The concentrations of produced proteins were measured in supernatants 24 hr after the transfections using ELISA (Figure 2). The transfection of EPCs with ANG-1, VEGF-A, or SDF-1α mRNA resulted in significantly increased production of ANG-1 (112 ± 6.1 ng/mL), VEGF-A (142 ± 6.7 ng/mL), and SDF-1α (17 ± 1.4 ng/mL) compared to the cells incubated with medium or medium containing the transfection reagent (Figure 2A). The transfection of EPCs with the mRNA cocktail also resulted in significantly increased protein synthesis (Figure 2B), and approximately 70 ± 7.1 ng/mL ANG-1, 58 ± 2.7 ng/mL VEGF-A, and 8 ± 0.4 ng/mL SDF-1α were detected. Here, the amount of produced ANG-1 was 1.6-fold, VEGF-A 2.4-fold, and SDF-1α 2.1-fold less than after the transfection of EPCs with single mRNAs. In the used mRNA cocktail, the total amount of mRNAs was 2.9 μg. To test the influence of higher mRNA amounts on protein synthesis, the cells were transfected with each single mRNA and EGFP mRNA, which was used as a filler mRNA, to obtain a total mRNA amount of 2.9 μg. Especially, after the co-transfection of ANG-1 mRNA with EGFP mRNA, a significant reduction of ANG-1 protein amount could be detected (Figure S1). In the case of VEGF-A mRNA, no influence could be detected. A slightly higher amount of SDF-1α was detected after the simultaneous transfection of cells with SDF-1α and EGFP mRNA. Furthermore, double combinations of ANG-1, VEGF-A, and SDF-1α mRNA transfections were tested (Figure S2). Here, the simultaneous transfection of cells with ANG-1 and VEGF-A or SDF-1α mRNA also resulted in a reduction in ANG-1 protein expression. These results indicate that the higher mRNA amount has an influence on ANG-1 production. The influence of mRNA transfections on the viability of EPCs was analyzed using PrestoBlue cell viability assay. 1 × 105 EPCs were cultivated overnight and transfected with 1.6 μg ANG-1, 0.8 μg VEGF-A, or 0.5 μg SDF-1α mRNA or with a triple mRNA cocktail. As a control, cells were treated with Opti-MEM containing the maximal amount of Lipofectamine 2000 (4 μL), which was used for the generation of transfection complexes. The viability of cells incubated with Opti-MEM (medium) was set to 100%, and the viability of samples was displayed relative to these cells. As shown in Figure 3, 24 hr post-transfection, the transfection of cells with synthetic mRNAs resulted in no significant differences in cell viability compared to the controls, cells incubated with medium or medium containing transfection reagent. The chemotactic activity of mRNA-engineered EPCs was determined using a migration assay. Transwell inserts seeded with 5 × 104 untreated EPCs were placed in wells containing 1 × 105 mRNA-transfected or untransfected EPCs. After 6 hr, the migrated cells were stained with 1 μg/mL DAPI and counted. The mRNA-transfected EPCs were able to attract significantly more EPCs compared to EPCs without mRNA transfection (ANG-1 mRNA, 405 ± 36 cells; VEGF-A mRNA, 426 ± 41 cells; SDF-1α mRNA, 400 ± 12 cells; and mRNA cocktail, 464 ± 27 cells versus EPCs without mRNA transfection [medium], 200 ± 10 cells; Figure 4). Compared to single-mRNA-transfected EPCs, the transfection of EPCs with the mRNA cocktail resulted in a slight increase of migrated cell numbers; however, this increase was not statistically significant. The treatment of cells with the transfection reagent also had no statistically significant influence on the migration behavior compared to the cells treated with medium. Interestingly, all EPCs transfected with a single mRNA or with the mRNA cocktail showed comparable migration activity. Overall, these results demonstrated an improvement of EPC migration toward EPCs transfected with proangiogenic mRNAs. The in vitro wound-healing assay, which mimics the in vivo wound-healing process, was used to analyze the migration behavior of mRNA-engineered EPCs growing in a monolayer culture. Therefore, 28,000 EPCs with or without mRNA transfection were seeded in each chamber of Culture-Insert 3 wells. After reaching confluence, the culture inserts were removed from the dish to generate an open wound field. To quantify the closed wound area, pictures were taken immediately after removing the insert (0 hr) and after 12, 24, and 36 hr. After 36 hr, the wound areas were closed for each sample (Figure 5). EPCs transfected with ANG-1 mRNA showed fastest closure of the wound area, with 94% already after 12 hr. In comparison, a wound closure area of about 45% was detected with EPCs treated only with medium and 38% with EPCs treated with the medium containing transfection reagent. After 24 hr, 100% of the wound area was closed in ANG-1 mRNA-transfected EPCs and 93% wound area was closed in SDF-1α mRNA-transfected EPCs. The closure of the wound area was significantly higher in these EPCs compared to the medium control. Especially, the transfection of EPCs with ANG-1 mRNA accelerated the migration process so that the wound area was completely closed already after 12 hr. In comparison, EPCs transfected with the mRNA cocktail showed significantly less wound closure area. Furthermore, the treatment of the cells with the transfection reagent had no significant influence on the migration and the speed of wound closure compared to untreated cells (medium control). The tube formation assay was performed to assess the angiogenic potential of mRNA-engineered EPCs in vitro. Therefore, 1 × 104 EPCs transfected with 1.6 μg ANG-1, 0.8 μg VEGF-A, or 0.5 μg SDF-1α mRNA or with a triple mRNA cocktail were seeded on Matrigel. Already within 4 hr, EPCs spontaneously initiated vascular morphogenesis and formed multicellular tubular networks (Figure 6). Using Angiogenesis Analyzer ImageJ plugin, the number of segments (elements, which are delimited by two junctions), number of master segments (segments, which are none exclusively implicated with one branch), number of nodes (pixels with at least 3 neighboring elements, corresponding to a bifurcation), total segment length (sum of lengths of the segments in the analyzed area), the total mesh areas (sum of areas enclosed by the segments or master segments), and the branching interval (the mean distance separating two branches [total segment length/number of branches]) were obtained. The unit of area and length is the pixel (px). EPCs engineered to simultaneously produce ANG-1, VEGF-A, and SDF-1α demonstrated highly increased network formation, compared to the untreated EPCs (medium). Thereby, the networks formed by EPCs transfected with the mRNA cocktail displayed higher numbers of nodes (545 ± 29 versus 205 ± 52), segments (185 ± 14 versus 52 ± 20), and master segments (110 ± 12 versus 24 ± 10), with an increase in total segment length (11,343 ± 660 px versus 3,786 ± 1750 px), total mesh area (569,094 ± 47,325 px versus 36,107 ± 24,228 px), as well as branching interval (116,306 ± 12,322 px versus 49,573 ± 21,438 px). The single transfection of EPCs either with ANG-1 or SDF-1α mRNA also led to an increase of parameters related to network formation. However, the increase was in the case of SDF-1α mRNA-transfected EPCs, statistically significant for number of nodes, segments, and total segment length, and in the case of ANG-1 mRNA-transfected EPCs, statistically significant for number of segments. In the case of VEGF-A, no increase could be detected compared to untreated EPCs. The overall results demonstrated an improvement of network formation characteristics in triple-mRNA-transfected EPCs, which led to a denser, highly branched tubular network with increased numbers of tubes, suggesting an improved induction of angiogenesis. The in vivo angiogenic potential of mRNA-engineered EPCs was analyzed using the CAM assay. Therefore, 1 × 105 EPCs were seeded and transfected the next day with 1.6 μg ANG-1, 0.8 μg VEGF-A, or 0.5 μg SDF-1α mRNA or with a triple mRNA cocktail. After 24 hr, cells were detached, and 4 × 105 cells were mixed in Matrigel and placed within a silicone ring on the CAM. As controls, EPCs incubated with medium or medium containing transfection reagent were also applied to the CAMs. For evaluation and quantification of angiogenesis, the area of sample application (within the Ø 0.8-mm silicone ring) of the fixed CAMs was photographed and analyzed using Wimasis WimCAM web-based service (Figure 7). Compared to the mRNA untreated cells (medium), EPCs transfected with the ANG-1 mRNA resulted in an augmented angiogenesis/vascularization determined by the significant increase in numbers of total branching points (701 ± 85 versus 337 ± 37), a higher vessel density (44.5% ± 0.6% versus 32% ± 2%), and increases in total segments (1,285 ± 195 px versus 659 ± 96 px) and total vessel network length (73,101 ± 5,986 px versus 46,551 ± 6908 px) in the analyzed area. Although EPCs transfected with the mRNA cocktail also showed an improvement of parameters regarding network formation on CAMs, the differences were not significantly different from untransfected EPCs. EPCs treated with only SDF-1α mRNA or VEGF-A showed no improved effect on the formation of new vasculature compared to the controls. The improvement of angiogenic potential of EPCs by the use of synthetic modified mRNA represents a promising strategy for the revascularization of ischemic tissues. In this study, we transiently modified murine EPCs with synthetic mRNAs encoding ANG-1, VEGF-A, and SDF-1α to augment the proangiogenic potential. Thereby, tissue repair and regeneration can be improved in the affected regions, first by the local production of growth factors improv" @default.
• W2891526049 created "2018-09-27" @default.
• W2891526049 creator A5003397453 @default.
• W2891526049 creator A5012215981 @default.
• W2891526049 creator A5023919458 @default.
• W2891526049 creator A5040149590 @default.
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• W2891526049 date "2018-12-01" @default.
• W2891526049 modified "2023-10-14" @default.
• W2891526049 title "Improving the Angiogenic Potential of EPCs via Engineering with Synthetic Modified mRNAs" @default.
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