Skip to main content

Advertisement

SpringerLink
Log in

Low levels of physiological interstitial flow eliminate morphogen gradients and guide angiogenesis

  • Original Paper
  • Published:
Angiogenesis Aims and scope Submit manuscript

Abstract

Convective transport can significantly distort spatial concentration gradients. Interstitial flow is ubiquitous throughout living tissue, but our understanding of how interstitial flow affects concentration gradients in biological processes is limited. Interstitial flow is of particular interest for angiogenesis because pathological and physiological angiogenesis is associated with altered interstitial flow, and both interstitial flow and morphogen gradients (e.g., vascular endothelial growth factor, VEGF) can potentially stimulate and guide new blood vessel growth. We designed an in vitro microfluidic platform to simulate 3D angiogenesis in a tissue microenvironment that precisely controls interstitial flow and spatial morphogen gradients. The microvascular tissue was developed from endothelial colony forming cell-derived endothelial cells extracted from cord blood and stromal fibroblasts in a fibrin extracellular matrix. Pressure in the microfluidic lines was manipulated to control the interstitial flow. A mathematical model of mass and momentum transport, and experimental studies with fluorescently labeled dextran were performed to validate the platform. Our data demonstrate that at physiological interstitial flow (0.1–10 μm/s), morphogen gradients were eliminated within hours, and angiogenesis demonstrated a striking bias in the opposite direction of interstitial flow. The interstitial flow-directed angiogenesis was dependent on the presence of VEGF, and the effect was mediated by αvβ3 integrin. We conclude that under physiological conditions, growth factors such as VEGF and fluid forces work together to initiate and spatially guide angiogenesis.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Carmeliet P, Jain RK (2000) Angiogenesis in cancer and other diseases. Nature 407(6801):249–257. doi:10.1038/35025220

    Article  CAS  PubMed  Google Scholar 

  2. Khurana R, Simons M, Martin JF, Zachary IC (2005) Role of angiogenesis in cardiovascular disease: a critical appraisal. Circulation 112(12):1813–1824. doi:10.1161/CIRCULATIONAHA.105.535294

    Article  PubMed  Google Scholar 

  3. Swartz MA, Lund AW (2012) Lymphatic and interstitial flow in the tumour microenvironment: linking mechanobiology with immunity. Nat Rev Cancer 12(3):210–219. doi:10.1038/nrc3186

    Article  CAS  PubMed  Google Scholar 

  4. Roussos ET, Condeelis JS, Patsialou A (2011) Chemotaxis in cancer. Nat Rev Cancer 11(8):573–587. doi:10.1038/nrc3078

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Wiig H, Swartz MA (2012) Interstitial fluid and lymph formation and transport: physiological regulation and roles in inflammation and cancer. Physiol Rev 92(3):1005–1060. doi:10.1152/physrev.00037.2011

    Article  CAS  PubMed  Google Scholar 

  6. Song JW, Munn LL (2011) Fluid forces control endothelial sprouting. Proc Natl Acad Sci USA 108(37):15342–15347. doi:10.1073/pnas.1105316108

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Polacheck WJ, German AE, Mammoto A, Ingber DE, Kamm RD (2014) Mechanotransduction of fluid stresses governs 3D cell migration. Proc Natl Acad Sci USA 111(7):2447–2452. doi:10.1073/pnas.1316848111

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Shi ZD, Tarbell JM (2011) Fluid flow mechanotransduction in vascular smooth muscle cells and fibroblasts. Ann Biomed Eng 39(6):1608–1619. doi:10.1007/s10439-011-0309-2

    Article  PubMed  PubMed Central  Google Scholar 

  9. Goel HL, Mercurio AM (2013) VEGF targets the tumour cell. Nat Rev Cancer 13(12):871–882. doi:10.1038/nrc3627

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Turner N, Grose R (2010) Fibroblast growth factor signalling: from development to cancer. Nat Rev Cancer 10(2):116–129. doi:10.1038/nrc2780

    Article  CAS  PubMed  Google Scholar 

  11. Jain RK, Schlenger K, Hockel M, Yuan F (1997) Quantitative angiogenesis assays: progress and problems. Nat Med 3(11):1203–1208

    Article  CAS  PubMed  Google Scholar 

  12. Bischel LL, Young EW, Mader BR, Beebe DJ (2013) Tubeless microfluidic angiogenesis assay with three-dimensional endothelial-lined microvessels. Biomaterials 34(5):1471–1477. doi:10.1016/j.biomaterials.2012.11.005

    Article  CAS  PubMed  Google Scholar 

  13. Zheng Y, Chen J, Craven M, Choi NW, Totorica S, Diaz-Santana A, Kermani P, Hempstead B, Fischbach-Teschl C, Lopez JA, Stroock AD (2012) In vitro microvessels for the study of angiogenesis and thrombosis. Proc Natl Acad Sci USA 109(24):9342–9347. doi:10.1073/pnas.1201240109

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Shields JD, Fleury ME, Yong C, Tomei AA, Randolph GJ, Swartz MA (2007) Autologous chemotaxis as a mechanism of tumor cell homing to lymphatics via interstitial flow and autocrine CCR7 signaling. Cancer Cell 11(6):526–538. doi:10.1016/j.ccr.2007.04.020

    Article  CAS  PubMed  Google Scholar 

  15. Alonzo LF, Moya ML, Shirure VS, George SC (2015) Microfluidic device to control interstitial flow-mediated homotypic and heterotypic cellular communication. Lab Chip 15(17):3521–3529

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Hsu YH, Moya ML, Abiri P, Hughes CC, George SC, Lee AP (2013) Full range physiological mass transport control in 3D tissue cultures. Lab Chip 13(1):81–89. doi:10.1039/c2lc40787f

    Article  CAS  PubMed  Google Scholar 

  17. Moya ML, Hsu YH, Lee AP, Hughes CC, George SC (2013) In vitro perfused human capillary networks. Tissue Eng Part C Methods 19(9):730–737. doi:10.1089/ten.TEC.2012.0430

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Melero-Martin JM, Khan ZA, Picard A, Wu X, Paruchuri S, Bischoff J (2007) In vivo vasculogenic potential of human blood-derived endothelial progenitor cells. Blood 109(11):4761–4768. doi:10.1182/blood-2006-12-062471

    Article  CAS  PubMed  Google Scholar 

  19. Chen X, Aledia AS, Popson SA, Him L, Hughes CC, George SC (2010) Rapid anastomosis of endothelial progenitor cell-derived vessels with host vasculature is promoted by a high density of cotransplanted fibroblasts. Tissue Eng Part A 16(2):585–594. doi:10.1089/ten.TEA.2009.0491

    Article  CAS  PubMed  Google Scholar 

  20. Luckett PM, Fischbarg J, Bhattacharya J, Silverstein SC (1989) Hydraulic conductivity of endothelial cell monolayers cultured on human amnion. Am J Physiol 256(6 Pt 2):H1675–H1683

    CAS  PubMed  Google Scholar 

  21. Nuttall AL (1987) Velocity of red blood cell flow in capillaries of the guinea pig cochlea. Hear Res 27(2):121–128

    Article  CAS  PubMed  Google Scholar 

  22. Ivanov KP, Kalinina MK, Levkovich YuI (1981) Blood flow velocity in capillaries of brain and muscles and its physiological significance. Microvasc Res 22(2):143–155

    Article  CAS  PubMed  Google Scholar 

  23. Swartz MA, Fleury ME (2007) Interstitial flow and its effects in soft tissues. Annu Rev Biomed Eng 9:229–256. doi:10.1146/annurev.bioeng.9.060906.151850

    Article  CAS  PubMed  Google Scholar 

  24. Jain RK, Stock RJ, Chary SR, Rueter M (1990) Convection and diffusion measurements using fluorescence recovery after photobleaching and video image analysis: in vitro calibration and assessment. Microvasc Res 39(1):77–93

    Article  CAS  PubMed  Google Scholar 

  25. Martino MM, Briquez PS, Ranga A, Lutolf MP, Hubbell JA (2013) Heparin-binding domain of fibrin(ogen) binds growth factors and promotes tissue repair when incorporated within a synthetic matrix. Proc Natl Acad Sci USA 110(12):4563–4568. doi:10.1073/pnas.1221602110

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Rutkowski JM, Swartz MA (2007) A driving force for change: interstitial flow as a morphoregulator. Trends Cell Biol 17(1):44–50. doi:10.1016/j.tcb.2006.11.007

    Article  CAS  PubMed  Google Scholar 

  27. Ghajar CM, Blevins KS, Hughes CC, George SC, Putnam AJ (2006) Mesenchymal stem cells enhance angiogenesis in mechanically viable prevascularized tissues via early matrix metalloproteinase upregulation. Tissue Eng 12(10):2875–2888. doi:10.1089/ten.2006.12.2875

    Article  CAS  PubMed  Google Scholar 

  28. Ghajar CM, Chen X, Harris JW, Suresh V, Hughes CC, Jeon NL, Putnam AJ, George SC (2008) The effect of matrix density on the regulation of 3-D capillary morphogenesis. Biophys J 94(5):1930–1941. doi:10.1529/biophysj.107.120774

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Brooks PC, Clark RA, Cheresh DA (1994) Requirement of vascular integrin alpha v beta 3 for angiogenesis. Science 264(5158):569–571

    Article  CAS  PubMed  Google Scholar 

  30. MacDonald TJ, Taga T, Shimada H, Tabrizi P, Zlokovic BV, Cheresh DA, Laug WE (2001) Preferential susceptibility of brain tumors to the antiangiogenic effects of an alpha(v) integrin antagonist. Neurosurgery 48(1):151–157

    CAS  PubMed  Google Scholar 

  31. Liu Z, Wang F, Chen X (2008) Integrin alpha(v)beta(3)-targeted cancer therapy. Drug Dev Res 69(6):329–339. doi:10.1002/ddr.20265

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Chen J, Green J, Yurdagul A Jr, Albert P, McInnis MC, Orr AW (2015) alphavbeta3 integrins mediate flow-induced NF-kappaB activation, proinflammatory gene expression, and early atherogenic inflammation. Am J Pathol 185(9):2575–2589. doi:10.1016/j.ajpath.2015.05.013

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Nisato RE, Tille JC, Jonczyk A, Goodman SL, Pepper MS (2003) alphav beta 3 and alphav beta 5 integrin antagonists inhibit angiogenesis in vitro. Angiogenesis 6(2):105–119. doi:10.1023/B:AGEN.0000011801.98187.f2

    Article  CAS  PubMed  Google Scholar 

  34. Hompland T, Ellingsen C, Ovrebo KM, Rofstad EK (2012) Interstitial fluid pressure and associated lymph node metastasis revealed in tumors by dynamic contrast-enhanced MRI. Cancer Res 72(19):4899–4908. doi:10.1158/0008-5472.CAN-12-0903

    Article  CAS  PubMed  Google Scholar 

  35. Chary SR, Jain RK (1989) Direct measurement of interstitial convection and diffusion of albumin in normal and neoplastic tissues by fluorescence photobleaching. Proc Natl Acad Sci USA 86(14):5385–5389

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Shin Y, Jeon JS, Han S, Jung GS, Shin S, Lee SH, Sudo R, Kamm RD, Chung S (2011) In vitro 3D collective sprouting angiogenesis under orchestrated ANG-1 and VEGF gradients. Lab Chip 11(13):2175–2181. doi:10.1039/c1lc20039a

    Article  CAS  PubMed  Google Scholar 

  37. Gerhardt H, Golding M, Fruttiger M, Ruhrberg C, Lundkvist A, Abramsson A, Jeltsch M, Mitchell C, Alitalo K, Shima D, Betsholtz C (2003) VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J Cell Biol 161(6):1163–1177. doi:10.1083/jcb.200302047

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Gerhardt H (2008) VEGF and endothelial guidance in angiogenic sprouting. Organogenesis 4(4):241–246

    Article  PubMed  PubMed Central  Google Scholar 

  39. Kim S, Lee H, Chung M, Jeon NL (2013) Engineering of functional, perfusable 3D microvascular networks on a chip. Lab Chip 13(8):1489–1500. doi:10.1039/c3lc41320a

    Article  CAS  PubMed  Google Scholar 

  40. Newman AC, Nakatsu MN, Chou W, Gershon PD, Hughes CC (2011) The requirement for fibroblasts in angiogenesis: fibroblast-derived matrix proteins are essential for endothelial cell lumen formation. Mol Biol Cell 22(20):3791–3800. doi:10.1091/mbc.E11-05-0393

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Lee H, Kim S, Chung M, Kim JH, Jeon NL (2014) A bioengineered array of 3D microvessels for vascular permeability assay. Microvasc Res 91:90–98. doi:10.1016/j.mvr.2013.12.001

    Article  CAS  PubMed  Google Scholar 

  42. Sobrino A, Phan DT, Datta R, Wang X, Hachey SJ, Romero-Lopez M, Gratton E, Lee AP, George SC, Hughes CC (2016) 3D microtumors in vitro supported by perfused vascular networks. Sci Rep 6:31589. doi:10.1038/srep31589

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Phan DT, Wang X, Craver BM, Sobrino A, Zhao D, Chen JC, Lee LY, George SC, Lee AP, Hughes CC (2017) A vascularized and perfused organ-on-a-chip platform for large-scale drug screening applications. Lab Chip 17(3):511–520. doi:10.1039/c6lc01422d

    Article  CAS  PubMed  Google Scholar 

  44. Sobrino A, Phan DT, Wang X, Datta R, Hachey SJ, López MR, Gratton E, Lee AP, George SC, Hughes CCW (2016) A vascularized and perfused human micro-tumor platform for cancer drug screening. SC Rep (in press)

  45. Dreher MR, Liu W, Michelich CR, Dewhirst MW, Yuan F, Chilkoti A (2006) Tumor vascular permeability, accumulation, and penetration of macromolecular drug carriers. J Natl Cancer Inst 98(5):335–344. doi:10.1093/jnci/djj070

    Article  CAS  PubMed  Google Scholar 

  46. Nagy JA, Feng D, Vasile E, Wong WH, Shih SC, Dvorak AM, Dvorak HF (2006) Permeability properties of tumor surrogate blood vessels induced by VEGF-A. Lab Invest 86(8):767–780. doi:10.1038/labinvest.3700436

    CAS  PubMed  Google Scholar 

  47. Vickerman V, Blundo J, Chung S, Kamm R (2008) Design, fabrication and implementation of a novel multi-parameter control microfluidic platform for three-dimensional cell culture and real-time imaging. Lab Chip 8(9):1468–1477. doi:10.1039/b802395f

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Verbridge SS, Chakrabarti A, DelNero P, Kwee B, Varner JD, Stroock AD, Fischbach C (2013) Physicochemical regulation of endothelial sprouting in a 3D microfluidic angiogenesis model. J Biomed Mater Res A 101(10):2948–2956. doi:10.1002/jbm.a.34587

    Article  PubMed  PubMed Central  Google Scholar 

  49. Nguyen DH, Stapleton SC, Yang MT, Cha SS, Choi CK, Galie PA, Chen CS (2013) Biomimetic model to reconstitute angiogenic sprouting morphogenesis in vitro. Proc Natl Acad Sci USA 110(17):6712–6717. doi:10.1073/pnas.1221526110

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Chan JM, Zervantonakis IK, Rimchala T, Polacheck WJ, Whisler J, Kamm RD (2012) Engineering of in vitro 3D capillary beds by self-directed angiogenic sprouting. PLoS ONE 7(12):e50582. doi:10.1371/journal.pone.0050582

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Li L, Wang L, Zhang W, Tang B, Zhang J, Song H, Yao D, Tang Y, Chen X, Yang Z, Wang G, Li X, Zhao J, Ding H, Reed E, Li QQ (2004) Correlation of serum VEGF levels with clinical stage, therapy efficacy, tumor metastasis and patient survival in ovarian cancer. Anticancer Res 24(3b):1973–1979

    CAS  PubMed  Google Scholar 

  52. Galie PA, Nguyen DH, Choi CK, Cohen DM, Janmey PA, Chen CS (2014) Fluid shear stress threshold regulates angiogenic sprouting. Proc Natl Acad Sci USA 111(22):7968–7973. doi:10.1073/pnas.1310842111

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Vickerman V, Kamm RD (2012) Mechanism of a flow-gated angiogenesis switch: early signaling events at cell-matrix and cell-cell junctions. Integr Biol (Camb) 4(8):863–874. doi:10.1039/c2ib00184e

    Article  CAS  Google Scholar 

  54. Helm CLE, Fleury ME, Zisch AH, Boschetti F, Swartz MA (2005) Synergy between interstitial flow and VEGF directs capillary morphogenesis in vitro through a gradient amplification mechanism. Proc Natl Acad Sci USA 102(44):15779–15784. doi:10.1073/pnas.0503681102

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Katsumi A, Orr AW, Tzima E, Schwartz MA (2004) Integrins in mechanotransduction. J Biol Chem 279(13):12001–12004. doi:10.1074/jbc.R300038200

    Article  CAS  PubMed  Google Scholar 

  56. Shamloo A, Xu H, Heilshorn S (2012) Mechanisms of vascular endothelial growth factor-induced pathfinding by endothelial sprouts in biomaterials. Tissue Eng Part A 18(3–4):320–330. doi:10.1089/ten.TEA.2011.0323

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the National Institutes of Health (UH3 TR00048, R01 CA170879, and F31 CA163049). We would like to thank Mr Vinson Tran, Mo Kebaili, Linda McCarthy (University of California, Irvine) and Sandra Lam (Washington University in St. Louis) for technical assistance. We would also like to thank Dr. Abraham Lee for helpful advice and material assistance during the development stages of this project.

Author information

Authors and Affiliations

  1. Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO, 63130, USA

    Venktesh S. Shirure, Andrew Lezia, Arnold Tao & Steven C. George

  2. Department of Biomedical Engineering, University of California, Irvine, CA, 92697, USA

    Luis F. Alonzo

  3. Department of Energy, Environment, and Chemical Engineering, Washington University in St. Louis, St. Louis, MO, 63130, USA

    Steven C. George

Authors
  1. Venktesh S. Shirure
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andrew Lezia
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Arnold Tao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Luis F. Alonzo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Steven C. George
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Steven C. George.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 15 kb)

Supplementary material 2 (PPTX 11692 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Shirure, V.S., Lezia, A., Tao, A. et al. Low levels of physiological interstitial flow eliminate morphogen gradients and guide angiogenesis. Angiogenesis 20, 493–504 (2017). https://doi.org/10.1007/s10456-017-9559-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10456-017-9559-4

Keywords

Search

Navigation