Advertisement

CD140b (PDGFRβ) Signaling in Adipose-Derived Stem Cells Mediates Angiogenic Behavior of Retinal Endothelial Cells

  • Ramesh Periasamy
  • Sally L. Elshaer
  • Rajashekhar GangarajuEmail author
Article
  • 57 Downloads
Part of the following topical collections:
  1. Invited Papers— Tao L. Lowe

Abstract

Adipose-derived stem cells (ASCs) are multipotent mesenchymal progenitor cells that have functional and phenotypic overlap with pericytes lining microvessels in adipose tissue. The role of CD140b [platelet-derived growth factor receptor-β (PDGFR-β)], a constitutive marker expressed by ASCs, in the angiogenic behavior of human retinal endothelial cells (HREs) is not known. CD140b was knocked down in ASCs using targeted siRNA and Lipofectamine transfection protocol. Both CD140b+ and CD140b− ASCs were tested for their proliferation (WST-1 reagent), adhesion (laminin-1-coated plates), and migration (wound-scratch assay). Angiogenic effect of CD140b+ and CD140b− ASCs on HREs was examined by co-culturing ASCs:HREs in 12:1 ratio for 6 days followed by visualization of vascular network by isolectin B4 staining. The RayBio® Membrane-Based Antibody Array was used to assess differences in human cytokines released by CD140b+ or CD140b− ASCs. Knockdown of CD140b in ASCs resulted in a significant 50% decrease in proliferation rate, 25% decrease in adhesion ability to laminin-1, and 50% decrease in migration rate, as compared to CD140b+ ASCs. Direct contact of ASCs expressing CD140b+ with HREs resulted in robust vascular network formation that was significantly reduced with using CD140b− ASCs. Of the 80 proteins tested, 45 proteins remained unchanged (> 0.5-–< 1.5-fold), 6 proteins including IL-10 were downregulated (< 0.5-fold), and 29 proteins including IL-16 and TNF-β were upregulated (> 1.5-fold) in CD140b− ASCs compared to CD140b+ ASCs. Our data demonstrate a substantial role for CD140b in the intrinsic abilities of ASCs and their angiogenic influence on HREs. Future studies are needed to fully explore the signaling of CD140b in ASCs in vivo for retinal regeneration.

Lay Summary

Adipose-derived stem cells (ASCs) obtained from human fat can differentiate into multiple tissues and also exhibit features of pericytes. In this study, we addressed the role of CD140b, a surface protein in ASCs that serves as a pericyte marker in angiogenic functioning of retinal endothelial cells. Our results demonstrate that CD140b is not only required for ASC survival but also mediates the production of certain paracrine factors that positively affect the angiogenic properties of retinal endothelial cells. Our study paves the way for future studies that are needed to fully explore CD140b signaling in ASCs in vivo for retinal regeneration.

Future Studies

Long-term studies are needed to study the safety and effectiveness of CD140b+ ASCs in retinal disease models to rule out any complications of stem cell treatment, including potential rejection and need for reinjection.

Keywords

Mesenchymal stem cells Pericyte Endothelial PDGF Migration Retina 

Notes

Acknowledgments

Authors wish to acknowledge Jack Anderson, BS, for technical support and Daniel Johnson, PhD, for the help with statistical analysis.

Author Contributions

Conceived and designed the experiments: RP, SLE, RG. Performed the experiments: RP, SLE. Analyzed the data: RP, SLE, RG. Wrote and reviewed the paper: RP, SLE, RG. Conceptualization and final approval: RG.

Funding Information

This study was funded by grants from National Eye Institute (EY023427) and unrestricted funds from Research to Prevent Blindness to R.G.

Compliance with Ethical Standards

Human ASC culture studies were approved for research per the University of Tennessee Institutional Biosafety and Institutional Review Board as exempt study.

Competing Interests

RG is a co-founder and hold equity in Cell Care Therapeutics Inc., whose interest is in the use of adipose-derived stromal cells in visual disorders. None of the other authors declare any financial conflicts.

Supplementary material

40883_2018_68_Fig5_ESM.png (1 mb)
Supplemental Figure 1

Flow cytometric characterization of cell surface proteins and transient transfection with CD140b siRNA in human ASCs. (A) Representative flow cytometric histograms of the expression of characteristic markers of CD105, CD31 and CD140b. Line shows discrimination of negative cells (isotype controls). (B) Mean expression of the surface markers from 3 representative human donors. (C) Relative gene expression of CD140b as normalized to β2-Micro globulin (BMG) internal control as measured by RT-PCR in ASCs lysates following transient transfection with siRNA against CD140b. (D) Flow cytometric analysis for surface expression of CD140b after transient transfection in ASCs. Data expressed as Mean ± SEM of n = 3 donors. *, p < 0.05. (PNG 1046 kb)

40883_2018_68_MOESM1_ESM.tif (36.3 mb)
High resolution image (TIF 37123 kb)
40883_2018_68_Fig6_ESM.png (357 kb)
Supplemental Figure 2

Multiple cytokine expression profile in the cell supernatants of ASCs. Data expressed as Mean of 8 donors. (PNG 357 kb)

40883_2018_68_MOESM2_ESM.tif (1.3 mb)
High resolution image (TIF 1297 kb)
40883_2018_68_Fig7_ESM.png (126 kb)
Supplemental Figure 3

Genetic deletion of CD140b does not alter proliferation of ASCs subjected to cellular stress. Proliferation of ASCs as measured by WST-1 cell proliferation reagent from cell subjected to (A) high glucose (B) oxidative stress (C) staurosporine and (D) Tumor Necrosis Factor alpha (TNF-α) cytokine. Data represent Mean ± SEM performed in duplicates. ***, p < 0.001; n = 3 donors. (PNG 126 kb)

40883_2018_68_MOESM3_ESM.tif (646 kb)
High resolution image (TIF 646 kb)

References

  1. 1.
    Cai X, Lin Y, Hauschka PV, Grottkau BE. Adipose stem cells originate from perivascular cells. Biol Cell. 2011;103(9):435–47.CrossRefGoogle Scholar
  2. 2.
    Tang W, Zeve D, Suh JM, Bosnakovski D, Kyba M, Hammer RE, et al. White fat progenitor cells reside in the adipose vasculature. Science. 2008;322(5901):583–6.CrossRefGoogle Scholar
  3. 3.
    Geevarghese A, Herman IM. Pericyte-endothelial crosstalk: implications and opportunities for advanced cellular therapies. Transl Res. 2014;163(4):296–306.CrossRefGoogle Scholar
  4. 4.
    da Silva Meirelles L, de Deus Wagatsuma VM, Malta TM, Bonini Palma PV, Araujo AG, Panepucci RA, et al. The gene expression profile of non-cultured, highly purified human adipose tissue pericytes: transcriptomic evidence that pericytes are stem cells in human adipose tissue. Exp Cell Res. 2016;349(2):239–54.CrossRefGoogle Scholar
  5. 5.
    Bunnell BA, Flaat M, Gagliardi C, Patel B, Ripoll C. Adipose-derived stem cells: isolation, expansion and differentiation. Methods. 2008;45(2):115–20.CrossRefGoogle Scholar
  6. 6.
    Frese L, Dijkman PE, Hoerstrup SP. Adipose tissue-derived stem cells in regenerative medicine. Transfusion Medicine and Hemotherapy: offizielles Organ der Deutschen Gesellschaft fur Transfusionsmedizin und Immunhamatologie. 2016;43(4):268–74.CrossRefGoogle Scholar
  7. 7.
    Bray GA. Medical consequences of obesity. J Clin Endocrinol Metab. 2004;89(6):2583–9.CrossRefGoogle Scholar
  8. 8.
    Rajashekhar G, Ramadan A, Abburi C, Callaghan B, Traktuev DO, Evans-Molina C, et al. Regenerative therapeutic potential of adipose stromal cells in early stage diabetic retinopathy. PLoS One. 2014;9(1):e84671.CrossRefGoogle Scholar
  9. 9.
    Sorrell JM, Baber MA, Traktuev DO, March KL, Caplan AI. The creation of an in vitro adipose tissue that contains a vascular-adipocyte complex. Biomaterials. 2011;32(36):9667–76.CrossRefGoogle Scholar
  10. 10.
    Verseijden F, Posthumus-van Sluijs SJ, Pavljasevic P, Hofer SO, van Osch GJ, Farrell E. Adult human bone marrow- and adipose tissue-derived stromal cells support the formation of prevascular-like structures from endothelial cells in vitro. Tissue Eng Part A. 2010;16(1):101–14.CrossRefGoogle Scholar
  11. 11.
    Strassburg S, Nienhueser H, Bjorn Stark G, Finkenzeller G, Torio-Padron N. Co-culture of adipose-derived stem cells and endothelial cells in fibrin induces angiogenesis and vasculogenesis in a chorioallantoic membrane model. J Tissue Eng Regen Med. 2016;10(6):496–506.CrossRefGoogle Scholar
  12. 12.
    Falcon BL, Swearingen M, Gough WH, Lee L, Foreman R, Uhlik M, et al. An in vitro cord formation assay identifies unique vascular phenotypes associated with angiogenic growth factors. PLoS One. 2014;9(9):e106901.CrossRefGoogle Scholar
  13. 13.
    Traktuev DO, Merfeld-Clauss S, Li J, Kolonin M, Arap W, Pasqualini R, et al. A population of multipotent CD34-positive adipose stromal cells share pericyte and mesenchymal surface markers, reside in a periendothelial location, and stabilize endothelial networks. Circ Res. 2008;102(1):77–85.CrossRefGoogle Scholar
  14. 14.
    Rehman J, Traktuev D, Li J, Merfeld-Clauss S, Temm-Grove CJ, Bovenkerk JE, et al. Secretion of angiogenic and antiapoptotic factors by human adipose stromal cells. Circulation. 2004;109(10):1292–8.CrossRefGoogle Scholar
  15. 15.
    Merfeld-Clauss S, Gollahalli N, March KL, Traktuev DO. Adipose tissue progenitor cells directly interact with endothelial cells to induce vascular network formation. Tissue Eng Part A. 2010;16(9):2953–66.CrossRefGoogle Scholar
  16. 16.
    Rivera JC, Dabouz R, Noueihed B, Omri S, Tahiri H, Chemtob S. Ischemic retinopathies: oxidative stress and inflammation. Oxidative Med Cell Longev 2017;2017:3940241, 1, 16.Google Scholar
  17. 17.
    Rajashekhar G. Mesenchymal stem cells: new players in retinopathy therapy. Front Endocrinol (Lausanne). 2014;5:59.Google Scholar
  18. 18.
    Andrae J, Gallini R, Betsholtz C. Role of platelet-derived growth factors in physiology and medicine. Genes Dev. 2008;22(10):1276–312.CrossRefGoogle Scholar
  19. 19.
    Ryu YJ, Cho TJ, Lee DS, Choi JY, Cho J. Phenotypic characterization and in vivo localization of human adipose-derived mesenchymal stem cells. Mol Cells. 2013;35(6):557–64.CrossRefGoogle Scholar
  20. 20.
    Baek SJ, Kang SK, Ra JC. In vitro migration capacity of human adipose tissue-derived mesenchymal stem cells reflects their expression of receptors for chemokines and growth factors. Exp Mol Med. 2011;43(10):596–603.CrossRefGoogle Scholar
  21. 21.
    Bourin P, Bunnell BA, Casteilla L, Dominici M, Katz AJ, March KL, et al. Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells: a joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT). Cytotherapy. 2013;15(6):641–8.CrossRefGoogle Scholar
  22. 22.
    Crisan M, Corselli M, Chen WC, Peault B. Perivascular cells for regenerative medicine. J Cell Mol Med. 2012;16(12):2851–60.CrossRefGoogle Scholar
  23. 23.
    Raghavan SS, Woon CY, Kraus A, Megerle K, Pham H, Chang J. Optimization of human tendon tissue engineering: synergistic effects of growth factors for use in tendon scaffold repopulation. Plast Reconstr Surg. 2012;129(2):479–89.CrossRefGoogle Scholar
  24. 24.
    Kim JH, Park SG, Song SY, Kim JK, Sung JH. Reactive oxygen species-responsive miR-210 regulates proliferation and migration of adipose-derived stem cells via PTPN2. Cell Death Dis. 2013;4:e588.CrossRefGoogle Scholar
  25. 25.
    Hye Kim J, Gyu Park S, Kim WK, Song SU, Sung JH. Functional regulation of adipose-derived stem cells by PDGF-D. Stem Cells. 2015;33(2):542–56.CrossRefGoogle Scholar
  26. 26.
    Gehmert S, Gehmert S, Hidayat M, Sultan M, Berner A, Klein S, et al. Angiogenesis: the role of PDGF-BB on adipose-tissue derived stem cells (ASCs). Clin Hemorheol Microcirc. 2011;48(1):5–13.Google Scholar
  27. 27.
    Rajashekhar G, Traktuev DO, Roell WC, Johnstone BH, Merfeld-Clauss S, Van Natta B, et al. IFATS collection: adipose stromal cell differentiation is reduced by endothelial cell contact and paracrine communication: role of canonical Wnt signaling. Stem Cells. 2008;26(10):2674–81.CrossRefGoogle Scholar
  28. 28.
    Elshaer SL, Abdelsaid MA, Al-Azayzih A, Kumar P, Matragoon S, Nussbaum JJ, et al. Pronerve growth factor induces angiogenesis via activation of TrkA: possible role in proliferative diabetic retinopathy. J Diabetes Res. 2013;2013:432659.CrossRefGoogle Scholar
  29. 29.
    Maumus M, Peyrafitte JA, D'Angelo R, Fournier-Wirth C, Bouloumie A, Casteilla L, et al. Native human adipose stromal cells: localization, morphology and phenotype. International Journal of Obesity (2005). 2011;35(9):1141–1153.Google Scholar
  30. 30.
    Lindahl P, Hellstrom M, Kalen M, Karlsson L, Pekny M, Pekna M, et al. Paracrine PDGF-B/PDGF-Rbeta signaling controls mesangial cell development in kidney glomeruli. Development. 1998;125(17):3313–22.Google Scholar
  31. 31.
    Soriano P. Abnormal kidney development and hematological disorders in PDGF beta-receptor mutant mice. Genes Dev. 1994;8(16):1888–96.CrossRefGoogle Scholar
  32. 32.
    Leveen P, Pekny M, Gebre-Medhin S, Swolin B, Larsson E, Betsholtz C. Mice deficient for PDGF B show renal, cardiovascular, and hematological abnormalities. Genes Dev. 1994;8(16):1875–87.CrossRefGoogle Scholar
  33. 33.
    Battegay EJ, Rupp J, Iruela-Arispe L, Sage EH, Pech M. PDGF-BB modulates endothelial proliferation and angiogenesis in vitro via PDGF beta-receptors. J Cell Biol. 1994;125(4):917–28.CrossRefGoogle Scholar
  34. 34.
    Lindahl P, Johansson BR, Leveen P, Betsholtz C. Pericyte loss and microaneurysm formation in PDGF-B-deficient mice. Science. 1997;277(5323):242–5.CrossRefGoogle Scholar
  35. 35.
    Ball SG, Shuttleworth A, Kielty CM. Inhibition of platelet-derived growth factor receptor signaling regulates Oct4 and Nanog expression, cell shape, and mesenchymal stem cell potency. Stem Cells. 2012;30(3):548–60.CrossRefGoogle Scholar
  36. 36.
    Tan HB, Giannoudis PV, Boxall SA, McGonagle D, Jones E. The systemic influence of platelet-derived growth factors on bone marrow mesenchymal stem cells in fracture patients. BMC Med. 2015;13:6.CrossRefGoogle Scholar
  37. 37.
    Gharibi B, Hughes FJ. Effects of medium supplements on proliferation, differentiation potential, and in vitro expansion of mesenchymal stem cells. Stem Cells Transl Med. 2012;1(11):771–82.CrossRefGoogle Scholar
  38. 38.
    Pountos I, Georgouli T, Henshaw K, Bird H, Jones E, Giannoudis PV. The effect of bone morphogenetic protein-2, bone morphogenetic protein-7, parathyroid hormone, and platelet-derived growth factor on the proliferation and osteogenic differentiation of mesenchymal stem cells derived from osteoporotic bone. J Orthop Trauma. 2010;24(9):552–6.CrossRefGoogle Scholar
  39. 39.
    Ng F, Boucher S, Koh S, Sastry KS, Chase L, Lakshmipathy U, et al. PDGF, TGF-beta, and FGF signaling is important for differentiation and growth of mesenchymal stem cells (MSCs): transcriptional profiling can identify markers and signaling pathways important in differentiation of MSCs into adipogenic, chondrogenic, and osteogenic lineages. Blood. 2008;112(2):295–307.CrossRefGoogle Scholar
  40. 40.
    Gehmert S, Gehmert S, Prantl L, Vykoukal J, Alt E, Song YH. Breast cancer cells attract the migration of adipose tissue-derived stem cells via the PDGF-BB/PDGFR-beta signaling pathway. Biochem Biophys Res Commun. 2010;398(3):601–5.CrossRefGoogle Scholar
  41. 41.
    Tokunaga A, Oya T, Ishii Y, Motomura H, Nakamura C, Ishizawa S, et al. PDGF receptor beta is a potent regulator of mesenchymal stromal cell function. J Bone Miner Res. 2008;23(9):1519–28.CrossRefGoogle Scholar
  42. 42.
    Chabot V, Dromard C, Rico A, Langonne A, Gaillard J, Guilloton F, et al. Urokinase-type plasminogen activator receptor interaction with beta1 integrin is required for platelet-derived growth factor-AB-induced human mesenchymal stem/stromal cell migration. Stem Cell Res Ther. 2015;6:188.CrossRefGoogle Scholar
  43. 43.
    Traktuev DO, Prater DN, Merfeld-Clauss S, Sanjeevaiah AR, Saadatzadeh MR, Murphy M, et al. Robust functional vascular network formation in vivo by cooperation of adipose progenitor and endothelial cells. Circ Res. 2009;104(12):1410–20.CrossRefGoogle Scholar
  44. 44.
    Freiman A, Shandalov Y, Rozenfeld D, Shor E, Segal S, Ben-David D, et al. Adipose-derived endothelial and mesenchymal stem cells enhance vascular network formation on three-dimensional constructs in vitro. Stem Cell Res Ther. 2016;7:5.CrossRefGoogle Scholar
  45. 45.
    Mazo M, Cemborain A, Gavira JJ, Abizanda G, Arana M, Casado M, et al. Adipose stromal vascular fraction improves cardiac function in chronic myocardial infarction through differentiation and paracrine activity. Cell Transplant. 2012;21(5):1023–37.CrossRefGoogle Scholar
  46. 46.
    Cho YJ, Song HS, Bhang S, Lee S, Kang BG, Lee JC, et al. Therapeutic effects of human adipose stem cell-conditioned medium on stroke. J Neurosci Res. 2012;90(9):1794–802.CrossRefGoogle Scholar
  47. 47.
    Nakagami H, Maeda K, Morishita R, Iguchi S, Nishikawa T, Takami Y, et al. Novel autologous cell therapy in ischemic limb disease through growth factor secretion by cultured adipose tissue-derived stromal cells. Arterioscler Thromb Vasc Biol. 2005;25(12):2542–7.CrossRefGoogle Scholar
  48. 48.
    Motro B, Itin A, Sachs L, Keshet E. Pattern of interleukin 6 gene expression in vivo suggests a role for this cytokine in angiogenesis. Proc Natl Acad Sci U S A. 1990;87(8):3092–6.CrossRefGoogle Scholar
  49. 49.
    Huang SP, Wu MS, Shun CT, Wang HP, Lin MT, Kuo ML, et al. Interleukin-6 increases vascular endothelial growth factor and angiogenesis in gastric carcinoma. J Biomed Sci. 2004;11(4):517–27.CrossRefGoogle Scholar
  50. 50.
    Dace DS, Khan AA, Kelly J, Apte RS. Interleukin-10 promotes pathological angiogenesis by regulating macrophage response to hypoxia during development. PLoS One. 2008;3(10):e3381.CrossRefGoogle Scholar
  51. 51.
    Bae J, Park D, Lee YS, Jeoung D. Interleukin-2 promotes angiogenesis by activation of Akt and increase of ROS. J Microbiol Biotechnol. 2008;18(2):377–82.Google Scholar
  52. 52.
    Cross MJ, Claesson-Welsh L. FGF and VEGF function in angiogenesis: signalling pathways, biological responses and therapeutic inhibition. Trends Pharmacol Sci. 2001;22(4):201–7.CrossRefGoogle Scholar
  53. 53.
    Sainson RC, Johnston DA, Chu HC, Holderfield MT, Nakatsu MN, Crampton SP, et al. TNF primes endothelial cells for angiogenic sprouting by inducing a tip cell phenotype. Blood. 2008;111(10):4997–5007.CrossRefGoogle Scholar
  54. 54.
    Lopatina T, Bruno S, Tetta C, Kalinina N, Porta M, Camussi G. Platelet-derived growth factor regulates the secretion of extracellular vesicles by adipose mesenchymal stem cells and enhances their angiogenic potential. Cell Commun Signal. 2014;12:26.CrossRefGoogle Scholar

Copyright information

© The Regenerative Engineering Society 2018

Authors and Affiliations

  1. 1.Department of Ophthalmology, Hamilton Eye InstituteUniversity of Tennessee Health Sciences CenterMemphisUSA
  2. 2.Department of Anatomy and NeurobiologyUniversity of Tennessee Health Sciences CenterMemphisUSA

Personalised recommendations