Abstract
The development and maturation of the lymphatic vasculature are essential for organ function with disruption leading to severe phenotypes. For example, malfunction of cardiac lymphatics results in myocardial oedema, persistent inflammation and reduced cardiac output. Thus, it is important to study the process of cardiac lymphatic formation and growth from the early stages of fetal development to adulthood. In the murine heart the lymphatics continue to develop and expand postnatally with extensive growth and patterning occurring up to at least 2 weeks after birth. Here, we describe a protocol for whole-mount, multi-view imaging and quantification of lymphatic vessel parameters, including vessel junction number (i.e., branching density), vessel length, and number of vessel end points in the murine postnatal heart. This protocol is based on the use of reliable antibodies against key markers of lymphatic endothelial cells (LECs), specifically the glycoprotein lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), the vascular endothelial growth factor receptor 3 (VEGFR3; also known as Fms-related receptor tyrosine kinase 4, FLT4), the mucin-type protein podoplanin (PDPN), and the co-receptor neuropilin 2 (NRP2). For imaging and quantitative analysis of the sub-epicardial network in neonatal hearts, VEGFR3 was selected given its exclusive expression in the lymphatic endothelium. In addition to LECs, LYVE1 expression was detected in tissue-resident macrophages, PDPN in the epicardium, and NRP2 in the autonomic nervous system of the heart. Overall, we characterized the expression patterns of commonly used lymphatic markers in the context of the neonatal heart and provide an image analysis pipeline that can be adapted to study other organs and systems (e.g., blood vasculature and nerve system).
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Acknowledgments
K.K. is funded by the Wellcome Trust Foundation (4-year PhD studentship 215103/Z/18/Z). J.M.V. is supported by a British Heart Foundation Intermediate Basic Science Research Fellowship (FS/19/31/34158). P.R.R. is supported by a British Heart Foundation chair award (CH/11/1/28798) and programme grant (RG/08/003/25264). We thank the Micron Oxford Advanced Bioimaging Unit for access to and training in the use of light-sheet microscopy: https://micronoxford.com.
Competing Interests
P.R.R. is co-founder and equity holder in OxStem Cardio, an Oxford University spin-out that seeks to exploit therapeutic strategies stimulating endogenous repair in cardiovascular regenerative medicine. The other authors declare no competing interests.
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Klaourakis, K., Riley, P.R., Vieira, J.M. (2022). Quantitative Three-Dimensional Analysis of the Lymphatic Vasculature in the Postnatal Mouse Heart. In: Benest, A.V. (eds) Angiogenesis. Methods in Molecular Biology, vol 2441. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2059-5_13
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DOI: https://doi.org/10.1007/978-1-0716-2059-5_13
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