Drainage of fluorescent liposomes from the vitreous to cervical lymph nodes via conjunctival lymphatics.

The use of liposomes as carriers for the delivery of biologically active molecules into the eye is of major interest. Indeed, encapsulation of biologically active molecules in liposomes may increase their bioavailability and may induce a sustained release, thus avoiding repeated intraocular injections and reducing side effects. We describe here the fate of rhodamine-conjugated liposomes (Rh-Lip) injected into the vitreous of normal Lewis rats. Twenty-four hours after intravitreal injection fluorescent liposomes were detected in the vitreous, the inner layer of the retina and to a lesser extent in the anterior segment of the eye. In addition, numerous Rh-Lip were also observed in the episclera and conjunctival stroma, in conjunctival lymphatic vessels and cervical lymph nodes (LN) draining the conjunctiva and the eye. In the LN, Rh-Lip were taken up by resident macrophages adjacent to CD4+ and CD8+ T cells. Thus, intravitreal injection of anti-inflammatory drugs loaded in liposomes could modulate the ocular immune microenvironment. In addition the passage of drugs into the cervical LN could alter the immune status of these LN and contribute to the regulation of intraocular inflammation. Our results suggest that this phenomenon should be taken into account to design new therapies based on intraocular drug administration.

An exquisite cross-control mechanism among endothelial cell fate regulators directs the plasticity and heterogeneity of lymphatic endothelial cells.

Arteriovenous-lymphatic endothelial cell fates are specified by the master regulators, namely, Notch, COUP-TFII, and Prox1. Whereas Notch is expressed in the arteries and COUP-TFII in the veins, the lymphatics express all 3 cell fate regulators. Previous studies show that lymphatic endothelial cell (LEC) fate is highly plastic and reversible, raising a new concept that all 3 endothelial cell fates may co-reside in LECs and a subtle alteration can result in a reprogramming of LEC fate. We provide a molecular basis verifying this concept by identifying a cross-control mechanism among these cell fate regulators. We found that Notch signal down-regulates Prox1 and COUP-TFII through Hey1 and Hey2 and that activated Notch receptor suppresses the lymphatic phenotypes and induces the arterial cell fate. On the contrary, Prox1 and COUP-TFII attenuate vascular endothelial growth factor signaling, known to induce Notch, by repressing vascular endothelial growth factor receptor-2 and neuropilin-1. We show that previously reported podoplanin-based LEC heterogeneity is associated with differential expression of Notch1 in human cutaneous lymphatics. We propose that the expression of the 3 cell fate regulators is controlled by an exquisite feedback mechanism working in LECs and that LEC fate is a consequence of the Prox1-directed lymphatic equilibrium among the cell fate regulators.

Ocular and systemic bio-distribution of rhodamine-conjugated liposomes loaded with VIP injected into the vitreous of Lewis rats.

PURPOSE: Local delivery of therapeutic molecules encapsulated within liposomes is a promising method to treat ocular inflammation. The purpose of the present study was to define the biodistribution of rhodamine-conjugated liposomes loaded with vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide, following their intravitreal (IVT) injection in normal rats. METHODS: Healthy seven- to eight-week-old Lewis male rats were injected into the vitreous with empty rhodamine-conjugated liposomes (Rh-Lip) or with VIP-loaded Rh-Lip (VIP-Rh-Lip; 50 mM of lipids with an encapsulation efficiency of 3.0+/-0.4 mmol VIP/mol lipids). Twenty-four h after IVT injection, the eyes, the cervical, mesenteric, and inguinal lymph nodes (LN), and spleen were collected. The phenotype and distribution of cells internalizing Rh-Lip and VIP-Rh-Lip were studied. Determination of VIP expression in ocular tissues and lymphoid organs and interactions with T cells in cervical LN was performed on whole mounted tissues and frozen tissue sections by immunofluorescence and confocal microscopy. RESULTS: In the eye, 24 h following IVT injection, fluorescent liposomes (Rh-Lip and VIP-Rh-Lip) were detected mainly in the posterior segment of the eye (vitreous, inner layer of the retina) and to a lesser extent at the level of the iris root and ciliary body. Liposomes were internalized by activated retinal Müller glial cells, ocular tissue resident macrophages, and rare infiltrating activated macrophages. In addition, fluorescent liposomes were found in the episclera and conjunctiva where free VIP expression was also detected. In lymphoid organs, Rh-Lip and VIP-Rh-Lip were distributed almost exclusively in the cervical lymph nodes (LN) with only a few Rh-Lip-positive cells detected in the spleen and mesenteric LN and none in the inguinal LN. In the cervical LN, Rh-Lip were internalized by resident ED3-positive macrophages adjacent to CD4 and CD8-positive T lymphocytes. Some of these T lymphocytes in close contact with macrophages containing VIP-Rh-Lip expressed VIP. CONCLUSIONS: Liposomes are specifically internalized by retinal Müller glial cells and resident macrophages in the eye. A limited passage of fluorescent liposomes from the vitreous to the spleen via the conventional outflow pathway and the venous circulation was detected. The majority of fluorescent liposomes deposited in the conjunctiva following IVT injection reached the subcapsular sinus of the cervical LN via conjuntival lymphatics. In the cervical LN, Rh-Lip were internalized by resident subcapsular sinus macrophages adjacent to T lymphocytes. Detection of VIP in both macrophages and T cells in cervical LN suggests that IVT injection of VIP-Rh-Lip may increase ocular immune privilege by modulating the loco-regional immune environment. In conclusion, our observations suggest that IVT injection of VIP-loaded liposomes is a promising therapeutic strategy to dampen ocular inflammation by modulating macrophage and T cell activation mainly in the loco-regional immune system.

Angiopoietin 2 regulates the transformation and integrity of lymphatic endothelial cell junctions.

Primitive lymphatic vessels are remodeled into functionally specialized initial and collecting lymphatics during development. Lymphatic endothelial cell (LEC) junctions in initial lymphatics transform from a zipper-like to a button-like pattern during collecting vessel development, but what regulates this process is largely unknown. Angiopoietin 2 (Ang2) deficiency leads to abnormal lymphatic vessels. Here we found that an ANG2-blocking antibody inhibited embryonic lymphangiogenesis, whereas endothelium-specific ANG2 overexpression induced lymphatic hyperplasia. ANG2 inhibition blocked VE-cadherin phosphorylation at tyrosine residue 685 and the concomitant formation of button-like junctions in initial lymphatics. The defective junctions were associated with impaired lymph uptake. In collecting lymphatics, adherens junctions were disrupted, and the vessels leaked upon ANG2 blockade or gene deletion. ANG2 inhibition also suppressed the onset of lymphatic valve formation and subsequent valve maturation. These data identify ANG2 as the first essential regulator of the functionally important interendothelial cell-cell junctions that form during lymphatic development.

Organ-specific lymphangiectasia, arrested lymphatic sprouting, and maturation defects resulting from gene-targeting of the PI3K regulatory isoforms p85alpha, p55alpha, and p50alpha.

The phosphoinositide 3-kinase (PI3K) family has multiple vascular functions, but the specific regulatory isoform supporting lymphangiogenesis remains unidentified. Here, we report that deletion of the Pik3r1 gene, encoding the regulatory subunits p85alpha, p55alpha, and p50alpha impairs lymphatic sprouting and maturation, and causes abnormal lymphatic morphology, without major impact on blood vessels. Pik3r1 deletion had the most severe consequences among gut and diaphragm lymphatics, which share the retroperitoneal anlage, initially suggesting that the Pik3r1 role in this vasculature is anlage-dependent. However, whereas lymphatic sprouting toward the diaphragm was arrested, lymphatics invaded the gut, where remodeling and valve formation were impaired. Thus, cell-origin fails to explain the phenotype. Only the gut showed lymphangiectasia, lymphatic up-regulation of the transforming growth factor-beta co-receptor endoglin, and reduced levels of mature vascular endothelial growth factor-C protein. Our data suggest that Pik3r1 isoforms are required for distinct steps of embryonic lymphangiogenesis in different organ microenvironments, whereas they are largely dispensable for hemangiogenesis.