Article | Published:

Characterization of smooth muscle cells from human atherosclerotic lesions and their responses to Notch signaling

Abstract

Atherosclerosis is the most common cause of heart disease and stroke. The use of animal models has advanced our understanding of the molecular signaling that contributes to atherosclerosis. Further understanding of this degenerative process in humans will require human tissue. Plaque removed during endarterectomy procedures to relieve arterial obstructions is usually discarded, but can be an important source of diseased cells. Resected tissue from carotid and femoral endarterectomy procedures were compared with carotid arteries from donors with no known cardiovascular disease. Vascular smooth muscle cells (SMC) contribute to plaque formation and may determine susceptibility to rupture. Notch signaling is implicated in the progression of atherosclerosis, and plays a receptor-specific regulatory role in SMC. We defined protein localization of Notch2 and Notch3 within medial and plaque SMC using immunostaining, and compared Notch2 and Notch3 levels in total plaques with whole normal arteries using immunoblot. We successfully derived SMC populations from multiple endarterectomy specimens for molecular analysis. To better define the protein signature of diseased SMC, we utilized sequential window acquisition of all theoretical spectra (SWATH) proteomic analysis to compare normal carotid artery SMC with endarterectomy-derived SMC. Similarities in protein profile and differentiation markers validated the SMC identity of our explants. We identified a subset of differentially expressed proteins that are candidates as functional markers of diseased SMC. To understand how Notch signaling may affect diseased SMC, we performed Jagged1 stimulation of primary cultures. In populations that displayed significant growth, Jagged1 signaling through Notch2 suppressed proliferation; cultures with low growth potential were non-responsive to Jagged1. In addition, Jagged1 did not promote contractile smooth muscle actin nor have a significant effect on the mature differentiated phenotype. Thus, SMC derived from atherosclerotic lesions show distinct proteomic profiles and have altered Notch signaling in response to Jagged1 as a differentiation stimulus, compared with normal SMC.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Ethics declarations

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. 1.

    Benjamin EJ, Blaha MJ, Chiuve SE, et al. Heart disease and stroke statistics-2017 update: a report from the American Heart Association. Circulation. 2017;135:e146–603.

  2. 2.

    Brown RA, Shantsila E, Varma C, et al. Current understanding of atherogenesis. Am J Med. 2017;130:268–82.

  3. 3.

    Ross R, Agius L. The process of atherogenesis--cellular and molecular interaction: from experimental animal models to humans. Diabetologia. 1992;35:S34–40.

  4. 4.

    Gomez D, Owens GK. Smooth muscle cell phenotypic switching in atherosclerosis. Cardiovasc Res. 2012;95:156–64.

  5. 5.

    Shankman LS, Gomez D, Cherepanova OA, et al. KLF4-dependent phenotypic modulation of smooth muscle cells has a key role in atherosclerotic plaque pathogenesis. Nat Med. 2015;21:628–37.

  6. 6.

    Caolo V, Schulten HM, Zhuang ZW, et al. Soluble Jagged-1 inhibits neointima formation by attenuating Notch-Herp2 signaling. Arterioscler Thromb Vasc Biol. 2011;31:1059–65.

  7. 7.

    Li Y, Takeshita K, Liu PY, et al. Smooth muscle Notch1 mediates neointimal formation after vascular injury. Circulation. 2009;119:2686–92.

  8. 8.

    Sakata Y, Xiang F, Chen Z, et al. Transcription factor CHF1/Hey2 regulates neointimal formation in vivo and vascular smooth muscle proliferation and migration in vitro. Arterioscler Thromb Vasc Biol. 2004;24:2069–74.

  9. 9.

    Boucher JM, Harrington A, Rostama B, et al. A receptor-specific function for Notch2 in mediating vascular smooth muscle cell growth arrest through cyclin-dependent kinase inhibitor 1B. Circ Res. 2013;113:975–85.

  10. 10.

    Fung E, Tang SM, Canner JP, et al. Delta-like 4 induces notch signaling in macrophages: implications for inflammation. Circulation. 2007;115:2948–56.

  11. 11.

    Nakano T, Fukuda D, Koga J, et al. Delta-like ligand 4-Notch signaling in macrophage activation. Arterioscler Thromb Vasc Biol. 2016;36:2038–47.

  12. 12.

    Fukuda D, Aikawa E, Swirski FK, et al. Notch ligand delta-like 4 blockade attenuates atherosclerosis and metabolic disorders. Proc Natl Acad Sci USA. 2012;109:E1868–77.

  13. 13.

    Fukuda D, Aikawa M. Expanding role of delta-like 4 mediated notch signaling in cardiovascular and metabolic diseases. Circ J. 2013;77:2462–8.

  14. 14.

    Briot A, Civelek M, Seki A, et al. Endothelial NOTCH1 is suppressed by circulating lipids and antagonizes inflammation during atherosclerosis. J Exp Med. 2015;212:2147–63.

  15. 15.

    Rizzo P, Mele D, Caliceti C, et al. The role of Notch in the cardiovascular system: potential adverse effects of investigational notch inhibitors. Front Oncol. 2014;4:384.

  16. 16.

    Aquila G, Pannella M, Morelli MB, et al. The role of Notch pathway in cardiovascular diseases. Glob Cardiol Sci Pract. 2013;2013:364–71.

  17. 17.

    Gillet LC, Navarro P, Tate S, et al. Targeted data extraction of the MS/MS spectra generated by data-independent acquisition: a new concept for consistent and accurate proteome analysis. Mol Cell Proteom. 2012;11:O111 016717.

  18. 18.

    Mi H, Muruganujan A, Casagrande JT, et al. Large-scale gene function analysis with the PANTHER classification system. Nat Protoc. 2013;8:1551–66.

  19. 19.

    Miano JM. Vascular smooth muscle cell differentiation-2010. J Biomed Res. 2010;24:169–80.

  20. 20.

    Tang Y, Boucher JM, Liaw L. Histone deacetylase activity selectively regulates Notch-mediated smooth muscle differentiation in human vascular cells. J Am Heart Association. 2012; https://doi.org/10.1161/JAHA.112.000901.

  21. 21.

    Tang Y, Urs S, Boucher J, et al. Notch and transforming growth factor-beta (TGFbeta) signaling pathways cooperatively regulate vascular smooth muscle cell differentiation. J Biol Chem. 2010;285:17556–63.

  22. 22.

    Nus M, Martinez-Poveda B, MacGrogan D, et al. Endothelial Jag1-RBPJ signalling promotes inflammatory leucocyte recruitment and atherosclerosis. Cardiovasc Res. 2016;112:568–580.

  23. 23.

    Gamrekelashvili J, Limbourg FP. Rules of attraction - endothelial Notch signaling controls leukocyte homing in atherosclerosis via Vcam1. Cardiovasc Res. 2016;112:527–529.

  24. 24.

    Quillard T, Devalliere J, Coupel S, et al. Inflammation dysregulates Notch signaling in endothelial cells: implication of Notch2 and Notch4 to endothelial dysfunction. Biochem Pharmacol. 2010;80:2032–41.

  25. 25.

    Zhang Q, Wang C, Liu Z, et al. Notch signal suppresses Toll-like receptor-triggered inflammatory responses in macrophages by inhibiting extracellular signal-regulated kinase 1/2-mediated nuclear factor kappaB activation. J Biol Chem. 2012;287:6208–17.

  26. 26.

    Gonzalez MJ, Ruiz-Garcia A, Monsalve EM, et al. DLK1 is a novel inflammatory inhibitor which interferes with NOTCH1 signaling in TLR-activated murine macrophages. Eur J Immunol. 2015;45:2615–27.

  27. 27.

    Kimball AS, Joshi AD, Boniakowski AE, et al. Notch regulates macrophage-mediated inflammation in diabetic wound healing. Front Immunol. 2017;8:635.

  28. 28.

    Sweeney C, Morrow D, Birney YA, et al. Notch 1 and 3 receptor signaling modulates vascular smooth muscle cell growth, apoptosis, and migration via a CBF-1/RBP-Jk dependent pathway. Faseb J. 2004;18:1421–3.

  29. 29.

    Morrow D, Guha S, Sweeney C, et al. Notch and vascular smooth muscle cell phenotype. Circ Res. 2008;103:1370–82.

  30. 30.

    Pessi T, Viiri LE, Raitoharju E, et al. Interleukin-6 and microRNA profiles induced by oral bacteria in human atheroma derived and healthy smooth muscle cells. Springerplus. 2015;4:206.

  31. 31.

    Aird WC. Endothelial cell heterogeneity. Cold Spring Harb Perspect Med. 2012;2:a006429.

  32. 32.

    Regan ER, Aird WC. Dynamical systems approach to endothelial heterogeneity. Circ Res. 2012;111:110–30.

  33. 33.

    Yuan L, Chan GC, Beeler D, et al. A role of stochastic phenotype switching in generating mosaic endothelial cell heterogeneity. Nat Commun. 2016;7:10160.

  34. 34.

    Coen M, Marchetti G, Palagi PM, et al. Calmodulin expression distinguishes the smooth muscle cell population of human carotid plaque. Am J Pathol. 2013;183:996–1009.

  35. 35.

    Langley SR, Willeit K, Didangelos A, et al. Extracellular matrix proteomics identifies molecular signature of symptomatic carotid plaques. J Clin Invest. 2017;127:1546–60.

  36. 36.

    Frise E, Knoblich JA, Younger-Shepherd S, et al. The Drosophila Numb protein inhibits signaling of the Notch receptor during cell-cell interaction in sensory organ lineage. Proc Natl Acad Sci USA. 1996;93:11925–32.

  37. 37.

    Flores AN, McDermott N, Meunier A, et al. NUMB inhibition of NOTCH signalling as a therapeutic target in prostate cancer. Nat Rev Urol. 2014;11:499–507.

  38. 38.

    Andersson ER, Lendahl U. Therapeutic modulation of Notch signalling--are we there yet? Nat Rev Drug Discov. 2014;13:357–78.

  39. 39.

    Redmond EM, Guha S, Walls D, et al. Investigational Notch and Hedgehog inhibitors--therapies for cardiovascular disease. Expert Opin Investig Drugs. 2011;20:1649–64.

  40. 40.

    Feil S, Fehrenbacher B, Lukowski R, et al. Transdifferentiation of vascular smooth muscle cells to macrophage-like cells during atherogenesis. Circ Res. 2014;115:662–7.

  41. 41.

    Naik V, Leaf EM, Hu JH, et al. Sources of cells that contribute to atherosclerotic intimal calcification: an in vivo genetic fate mapping study. Cardiovasc Res. 2012;94:545–54.

  42. 42.

    Dikic I, Schmidt MH. Notch: implications of endogenous inhibitors for therapy. Bioessays. 2010;32:481–7.

Download references

Acknowledgements

This study was supported by NIH grant R01HL070865 to LL. Support for a research coordinator and procurement of human tissue was provided via a pilot project award from the Cardiovascular Research Institute at Maine Medical Center. Individuals who have greatly facilitated this project include MMC vascular surgeons Christopher Healey, Robert Hawkins, Elizabeth Blazick, and Paul Bloch, research coordinators Melissa Garrett and Dana Tripp, research assistant Debra Wright, and operating room nurse manager Cynthia Jones (all at Maine Medical Center). We thank Lauren Richey DVM, PhD (Tufts University) for assistance in histopathology of atherosclerotic plaques. Core facilities that assisted with tissue processing and histology (Grazina Mangoba and Mayasah Al Hashimi of the Histopathology and Histomorphometry Core) and mass spectrometry (Proteomics and Lipidomics Core Facility) were funded via NIH COBRE awards 8P30GM103392 (D. St. Germain, PI) and 1P20GM121301 (LL, PI). Partial support for the core facilities used in this research was provided by NIH grant U54GM115516 (C. Rosen, PI). JD-K was supported by fellowship 16PRE29870001 from the American Heart Association.

Author information

Conflict of interest

The authors declare that they have no conflict of interest.

Correspondence to Lucy Liaw.

Electronic supplementary material

Supplemental Figure 1

Supplemental Table 1

Supplemental Table 2

Supplemental Table 3

Supplemental Table 4

Supplemental Table 5

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Further reading

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7