Abstract
Microvascular endothelial cell-mural cell interactions are instrumental in modulating both physiological and pathologic angiogenesis. Pericyte-endothelial cell communication through direct physical associations and secreted effectors comprises a bidirectional signal array that regulates vascular maturation and integrity. As endothelial cell proliferation, migration, and morphogenesis are key elements of vascular growth and remodeling during angiogenesis, we have developed novel preclinical systems for studying the roles of endothelial-mural cell dynamics on cell cycle entry and angiogenic activity in vitro. These coculture models not only enable evaluation of endothelial cell-pericyte “cross talk” but also allow for the quantitative analysis of both heterotypic contact-dependent and contact-independent cell cycle progression in either cell population, as well as angiogenic sprouting in three-dimensional vascular networks. Cells actively proliferating in two-dimensional assays can be labeled via incorporation of 5-ethynyl-2′-deoxyuridine (EdU) into their DNA. Additionally, each cell population can be vitally labeled with a variety of cell-specific and/or membrane-permeant lipophilic dyes prior to coculture, such as DiO, or through immunofluorescence of mural or endothelial cell-specific markers after cellular fixation and/or permeabilization. Ultimately, this experimental approach can be used to investigate cellular contact-dependent and soluble mechanisms mediating mural-endothelial cell interactions, which may be instrumental in microvascular development and remodeling in vivo.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Conway EM, Collen D et al (2001) Molecular mechanisms of blood vessel growth. http://cardiovascres.oxfordjournals.org/content/49/3/507.short
Orlidge A, D’Amore PA (1987) Inhibition of capillary endothelial cell growth by pericytes and smooth muscle cells. J Cell Biol 105:1455–1462
Armulik A, Genové G, Mäe M, Nisancioglu M, Wallgard E, Niaudet C, He L, Norlin J, Lindblom P, Strittmatter K, Johansson B, Betsholtz C (2010) Pericytes regulate the blood-brain barrier. Nature 468:557–561
Hall CN, Reynell C, Gesslein B, Hamilton NB, Mishra A, Sutherland BA, O’Farrell FM, Buchan AM, Lauritzen M, Attwell D (2014) Capillary pericytes regulate cerebral blood flow in health and disease. Nature 508:55–60
Papetti M, Shujath J, Riley KN, Herman IM (2003) FGF-2 antagonizes the TGF-beta1-mediated induction of pericyte alpha-smooth muscle actin expression: a role for myf-5 and Smad-mediated signaling pathways. Invest Ophthalmol Vis Sci 44:4994–5005
Antonelli-Orlidge A, Saunders K, Smith S, D’Amore P (1989) An activated form of transforming growth factor beta is produced by cocultures of endothelial cells and pericytes. Proc Natl Acad Sci 86:4544–4548
Hellstrom M, Lindahl P, Abramsson A, Betsholtz C (1999) Role of PDGF-B and PDGFR-beta in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse. http://dev.biologists.org/content/126/14/3047.short
Greenberg JI, Shields DJ, Barillas SG, Acevedo LM, Murphy E, Huang J, Scheppke L, Stockmann C, Johnson RS, Angle N, Cheresh DA (2008) A role for VEGF as a negative regulator of pericyte function and vessel maturation. Nature 456:809–813
Wu D, Minami M, Kawamura H, Puro D (2006) Electrotonic transmission within pericyte‐containing retinal microvessels. Microcirculation 13:353–363
Gerhardt H, Wolburg H, Redies C (2000) N-cadherin mediates pericytic-endothelial interaction during brain angiogenesis in the chicken. Dev Dyn 218:472–479
Sainson RC, Harris AL (2008) Regulation of angiogenesis by homotypic and heterotypic notch signalling in endothelial cells and pericytes: from basic research to potential therapies. doi: 10.1007/s10456-008-9098-0
Geevarghese A, Herman IM (2014) Pericyte-endothelial crosstalk: implications and opportunities for advanced cellular therapies. Transl Res 163:296–306
Dulmovits BM, Herman IM (2012) Microvascular remodeling and wound healing: a role for pericytes. Int J Biochem Cell Biol 44:1800–1812
Kutcher ME, Herman IM (2009) The pericyte: cellular regulator of microvascular blood flow. Microvasc Res 77:235–246
Nayak RC, Herman IM (2001) Bovine retinal microvascular pericytes. http://link.springer.com/10.1385/1-59259-143-4:247
Herman IM, Leung A (2009) Creation of human skin equivalents for the in vitro study of angiogenesis in wound healing. doi: 10.1007/978-1-59745-241-0_14
Mendel TA, Clabough EB, Kao DS, Demidova-Rice TN, Durham JT, Zotter BC, Seaman SA, Cronk SM, Rakoczy EP, Katz AJ, Herman IM, Peirce SM, Yates PA (2013) Pericytes derived from adipose-derived stem cells protect against retinal vasculopathy. PLoS One 8:e65691
Frank RN, Dutta S, Mancini MA (1987) Pericyte coverage is greater in the retinal than in the cerebral capillaries of the rat. http://www.iovs.org/content/28/7/1086.short
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Sheets, A.R., Durham, J.T., Herman, I.M. (2016). Quantitative Imaging-Based Examination of Pericytes Controlling Endothelial Growth Dynamics and Angiogenesis. In: Martin, S., Hewett, P. (eds) Angiogenesis Protocols. Methods in Molecular Biology, vol 1430. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3628-1_15
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3628-1_15
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3626-7
Online ISBN: 978-1-4939-3628-1
eBook Packages: Springer Protocols