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

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.

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Change history

  • 24 February 2020

    An affiliation for Dr. Sally L. Elshaer was inadvertently omitted from this paper:

    Department of Pharmacology and Toxicology, College of Pharmacy, Mansoura University, 60 Elgomhoria St, Mansoura, 35516, Egypt

  • 24 February 2020

    An affiliation for Dr. Sally L. Elshaer was inadvertently omitted from this paper:

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.

    Google 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.

    CAS  Google Scholar 

  3. 3.

    Geevarghese A, Herman IM. Pericyte-endothelial crosstalk: implications and opportunities for advanced cellular therapies. Transl Res. 2014;163(4):296–306.

    Google 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.

    Google 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.

    CAS  Google 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.

    Google Scholar 

  7. 7.

    Bray GA. Medical consequences of obesity. J Clin Endocrinol Metab. 2004;89(6):2583–9.

    CAS  Google 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.

    Google 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.

    Google 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.

    CAS  Google 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.

    CAS  Google 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.

    Google 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.

    CAS  Google 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.

    Google 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.

    CAS  Google 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.

    CAS  Google 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.

    CAS  Google 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.

    CAS  Google 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.

    Google 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.

    CAS  Google 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.

    CAS  Google 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.

    CAS  Google 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.

    Google 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.

    Google 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.

    Google 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.

    CAS  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.

    CAS  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.

    CAS  Google 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.

    CAS  Google 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.

    CAS  Google 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.

    CAS  Google 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.

    CAS  Google 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.

    Google 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.

    CAS  Google 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.

    Google 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.

    CAS  Google 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.

    CAS  Google 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.

    CAS  Google 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.

    Google 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.

    CAS  Google 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.

    Google 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.

    Google 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.

    CAS  Google 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.

    CAS  Google 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.

    CAS  Google 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.

    CAS  Google 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.

    Google 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.

    CAS  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.

    CAS  Google 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.

    CAS  Google 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.

    Google Scholar 

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Acknowledgments

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

Funding

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

Author information

Affiliations

Authors

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.

Corresponding author

Correspondence to Rajashekhar Gangaraju.

Ethics declarations

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.

Electronic Supplementary Material

Supplemental Figure 1
figure5

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)

Supplemental Figure 2
figure6

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

Supplemental Figure 3
figure7

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)

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Periasamy, R., Elshaer, S.L. & Gangaraju, R. CD140b (PDGFRβ) Signaling in Adipose-Derived Stem Cells Mediates Angiogenic Behavior of Retinal Endothelial Cells. Regen. Eng. Transl. Med. 5, 1–9 (2019). https://doi.org/10.1007/s40883-018-0068-9

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Keywords

  • Mesenchymal stem cells
  • Pericyte
  • Endothelial
  • PDGF
  • Migration
  • Retina