The primary finding in this study is that lymphatic and blood endothelial cells make apparent contact at the capillary level in adult rat microvascular networks. Based on intravital microscopy (Clark and Clark, 1937
; Hauck, 1973
) and contrast filling studies from either the vascular or the lymphatic side (Schmid-Schönbein et al., 1977
; Skalak et al., 1984
; Unthank and Bohlen, 1988
), there is no evidence that the two systems form connections with an open lumen capable of permitting fluid exchange. Instead, our results suggest the existence of cellular contact between lymphatic and blood endothelial cells along blind-ended capillary vessels. We observe examples of lymphatic/blood capillary alignment in the whole mount preparations of ileum muscle coat and cremaster muscle, suggesting that lymphatic/blood vessel connections at the capillary level are not tissue-specific. Together with our observation of a subset of lymphatic blind-ended endothelial cell segments with similar endothelial cell structures associated with capillary sprouts and a lack of lymphatic marker expression emphasize the need to investigate the common cellular and molecular mechanisms involved in lymphatic and blood vessel growth and the functional significance of these cell connections.
Lymphatic/venous communication at the macrocirculation level outside the entry points to the subclavian and thoracic ducts have been previously reported especially in situations of increased pressure due to vessel occlusion (Miller, 1982
; Yoffrey and Courtice, 1970
). In the heart, lymphatic/venous anastomoses between vessels in the microcirculation with diameters 30 μm – 50 μm were observed at 7 and 14 days post lymphatic occlusion (Eliska and Eliskova, 1975
). In the context of these findings, our observations suggest that physical lymphatic/blood vessel connections at the capillary level are pre-established in unstimulated tissues.
Patent connections between the lymphatic and blood vessel systems in peripheral tissues have recently been proposed to exist in mice lacking certain signaling proteins. Intravenous injection of FITC-labeled dextran identified abnormal lymphatic/blood vessel lumenal connections in SLP-76−/−
mice (Abtahian et al., 2003
). In Fiaf−/−
(fasting-induced adipose factor) mice, intravenous injection of BSI lectin served to identify lymphatic/blood vessel connections during postnatal development (Backhed et al., 2007
). Fiaf is believed to be an upstream regulator of Prox-1. Johnson et al. (2008)
demonstrated that lymphatic endothelial cell identity depends on Prox-1 activity and further found that Prox-1 conditional mutants displayed lumenal connections between the lymphatic and blood vascular systems as determined by dextran infusion and the presence of blood-filled lymphatics. In the context of these studies, our observations suggest sites of pre-existing lymphatic/blood vessel connections at the capillary level that can be influenced by local epigenetic stimuli.
The presence of lymphatic/blood endothelial connections highlights the possibility for shared patterning signals between lymphatic and blood microvascular systems in the adult. Molecular players, such as vascular endothelial growth factors and angiopoietins, are involved in angiogenesis and play similar roles in lymphangiogenesis (Karpanen and Makinen, 2006
). Recently, macrophages have been implicated in both angiogenesis and lymphangiogenesis via paracrine mechanisms (El-Chemaly et al., 2009
; Maruyama et al., 2005
). During inflammation induced lymphangiogenesis, macrophages have also been shown to be able to differentiate into lymphatic endothelial cells (El-Chemaly et al., 2009
). Currently the role of macrophages at sites of apparent lymphatic/blood endothelial connections in our unstimulated tissues remains unclear (Online Supplemental Figure B
). Further investigation during inflammatory conditions regarding the mechanistic role of shared molecular and cellular players will provide valuable insight to the formation and functionality of these connections.
The observation of blood vessel-like lymphatic blind-ended segments originating from lymphatic capillaries supports the ability for cells of different phenotypes to interact with each other and therefore, the presence of direct contact lymphatic/blood endothelial cell connections. While a dynamic time-lapse study is required to confirm that the blind-ended vessels analyzed in this study are in fact sprouting, previous characterizations of angiogenesis in this rat mesenteric tissue suggest that these structures are indicative of a vessel growth process (Anderson et al., 2004
; Murfee et al., 2006
). Additionally, we observed a lack of evidence for apoptosis along blind-ended vessels in rat mesenteric tissue as determined by the TUNEL technique (data not shown). Initial lymphatics are generally considered to be void of vascular pericytes. Our observations of a subset of blind-ended lymphatic endothelial cell segments lacking lymphatic marker expression and able to recruit vascular pericytes highlight the potential for the local environment to influence endothelial cell plasticity.
In summary, our work provides anatomical evidence for cell-cell contact between lymphatic and blood vessels at the capillary level. While the functionality of these apparent connections during tissue remodeling inflammation and other remodeling scenarios requires further investigation, our work offers a novel perspective for understanding the common cell lineage and overlapping mechanisms associated with angiogenesis and lymphangiogenesis.