Invariant asymmetry renews the lymphatic vasculature during homeostasis.

BACKGROUND: The lymphatic vasculature regulates tissue physiology and immunity throughout life. The self renewal mechanism that maintains the lymphatic vasculature during conditions of homeostasis is unknown. The purpose of this study was to investigate the cellular mechanism of lymphatic endothelial cell (LEC) self renewal and lymphatic vessel maintenance. METHODS: Inductive genetic techniques were used to label LECs with tandem dimer tomato (tdT) in adult mice. Two types of studies were performed, those with high dose inductive conditions to label nearly all the lymphatic vessels and studies with low dose inductive conditions to stochastically label individual clones or small populations of LECs. We coupled image guidance techniques and live fluorescence microscopy imaging with lineage tracing to track the fate of entire tdT(+) cutaneous lymphatic vessels or the behavior of individual or small populations of LECs over 11 months. We tracked the fate of 110 LEC clones and 80 small LEC populations (clusters of 2-7 cells) over 11 months and analyzed their behavior using quantitative techniques. RESULTS: The results of the high dose inductive studies showed that the lymphatic vessels remained tdT(+) over 11 months, suggesting passage and expression of the tdT transgene from LEC precursors to progenies, an intrinsic model of self- renewal. Interestingly, the morphology of tdT(+) lymphatic vasculature appeared relatively stable without significant remodeling during this time period. By following the behavior of labeled LEC clones or small populations of LECs individually over 11 months, we identified diverse LEC fates of proliferation, quiescence, and extinction. Quantitative analysis of this data revealed that the average lymphatic endothelial clone or small population remained stable in size despite diverse individual fates. CONCLUSION: The results of these studies support a mechanism of invariant asymmetry to self renew the lymphatic vasculature during homeostasis. These original findings raise important questions related to the plasticity and self renewal properties that maintain the lymphatic vasculature during life.

Lymphatic endothelial lineage assemblage during corneal lymphangiogenesis.

Postnatal inflammatory lymphangiogenesis presumably requires precise regulatory processes to properly assemble proliferating lymphatic endothelial cells (LECs). The specific mechanisms that regulate the assembly of LECs during new lymphatic vessel synthesis are unclear. Dynamic endothelial shuffling and rearrangement has been proposed as a mechanism of blood vessel growth. We developed genetic lineage-tracing strategies using an inductive transgenic technology to track the fate of entire tandem dimer tomato-positive (tdT) lymphatic vessels or small, in some cases clonal, populations of LECs. We coupled this platform with a suture-induced mouse model of corneal lymphangiogenesis and used different analytic microscopy techniques including serial live imaging to study the spatial properties of proliferating tdT(+) LEC progenies. LEC precursors and their progeny expanded from the corneal limbal lymphatic vessel and were assembled contiguously to comprise a subunit within a new lymphatic vessel. VE-cadherin blockade induced morphologic abnormalities in newly synthesized lymphatic vessels, but did not disrupt the tdT(+) lymphatic endothelial lineage assembly. Analysis of this static and dynamic data based largely on direct in vivo observations supports a model of lymphatic endothelial lineage assemblage during corneal inflammatory lymphangiogenesis.

Nerve growth factor regulates neurolymphatic remodeling during corneal inflammation and resolution.

The cellular and physiologic mechanisms that regulate the resolution of inflammation remain poorly defined despite their widespread importance in improving inflammatory disease outcomes. We studied the resolution of two cardinal signs of inflammation-pain and swelling-by investigating molecular mechanisms that regulate neural and lymphatic vessel remodeling during the resolution of corneal inflammation. A mouse model of corneal inflammation and wound recovery was developed to study this process in vivo. Administration of nerve growth factor (NGF) increased pain sensation and inhibited neural remodeling and lymphatic vessel regression processes during wound recovery. A complementary in vivo approach, the corneal micropocket assay, revealed that NGF-laden pellets stimulated lymphangiogenesis and increased protein levels of VEGF-C. Adult human dermal lymphatic endothelial cells did not express canonical NGF receptors TrkA and p75NTR or activate downstream MAPK- or Akt-pathway effectors in the presence of NGF, although NGF treatment increased their migratory and tubulogenesis capacities in vitro. Blockade of the VEGF-R2/R3 signaling pathway ablated NGF-mediated lymphangiogenesis in vivo. These findings suggest a hierarchical relationship with NGF functioning upstream of the VEGF family members, particularly VEGF-C, to stimulate lymphangiogenesis. Taken together, these studies show that NGF stimulates lymphangiogenesis and that NGF may act as a pathogenic factor that negatively regulates the normal neural and lymphatic vascular remodeling events that accompany wound recovery.

Lymphatic vessel memory stimulated by recurrent inflammation.

Inflammation stimulates new lymphatic vessel growth (inflammatory lymphangiogenesis). One key question is how recurrent inflammation, a common clinical condition, regulates lymphatic vessel remodeling. We show here that recurrent inflammation accelerated the development a functional lymphatic vessel network. This observation suggests a novel program of lymphangiogenesis and identifies a property of lymphatic vessel memory in response to recurrent inflammation. A brief episode of initial inflammation regressed lymphatic vessels, and a significant increase in CD11b(+) macrophages were associated with the development of lymphatic vessel memory. These vessels had major differences in the structure and the spatial distribution of specialized lymphatic vessel features. Surprisingly, we found that the lymphatic vessel memory response did not depend on the vascular endothelial growth factor C or A pathway, indicating that different molecular pathways regulate inflammatory lymphangiogenesis and lymphatic vessel memory. These findings uncover a priming mechanism to facilitate a rapid lymphatic vessel memory response: a potential important component of peripheral host defense.