Lymphatic regulator PROX1 determines Schlemm's canal integrity and identity.
ABSTRACT: Schlemm's canal (SC) is a specialized vascular structure in the eye that functions to drain aqueous humor from the intraocular chamber into systemic circulation. Dysfunction of SC has been proposed to underlie increased aqueous humor outflow (AHO) resistance, which leads to elevated ocular pressure, a factor for glaucoma development in humans. Here, using lymphatic and blood vasculature reporter mice, we determined that SC, which originates from blood vessels during the postnatal period, acquires lymphatic identity through upregulation of prospero homeobox protein 1 (PROX1), the master regulator of lymphatic development. SC expressed lymphatic valve markers FOXC2 and integrin ?9 and exhibited continuous vascular endothelial-cadherin (VE-cadherin) junctions and basement membrane, similar to collecting lymphatics. SC notably lacked luminal valves and expression of the lymphatic endothelial cell markers podoplanin and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1). Using an ocular puncture model, we determined that reduced AHO altered the fate of SC both during development and under pathologic conditions; however, alteration of VEGF-C/VEGFR3 signaling did not modulate SC integrity and identity. Intriguingly, PROX1 expression levels linearly correlated with SC functionality. For example, PROX1 expression was reduced or undetectable under pathogenic conditions and in deteriorated SCs. Collectively, our data indicate that PROX1 is an accurate and reliable biosensor of SC integrity and identity.
Project description:Primary open-angle glaucoma (POAG) is often caused by elevated intraocular pressure (IOP), which arises due to increased resistance to aqueous humor outflow (AHO). Aqueous humor flows through Schlemm's canal (SC), a lymphatic-like vessel encircling the cornea, and via intercellular spaces of ciliary muscle cells. However, the mechanisms underlying increased AHO resistance are poorly understood. Here, we demonstrate that signaling between angiopoietin (Angpt) and the Angpt receptor Tie2, which is critical for SC formation, is also indispensable for maintaining SC integrity during adulthood. Deletion of Angpt1/Angpt2 or Tie2 in adult mice severely impaired SC integrity and transcytosis, leading to elevated IOP, retinal neuron damage, and impairment of retinal ganglion cell function, all hallmarks of POAG in humans. We found that SC integrity is maintained by interconnected and coordinated functions of Angpt-Tie2 signaling, AHO, and Prox1 activity. These functions diminish in the SC during aging, leading to impaired integrity and transcytosis. Intriguingly, Tie2 reactivation using a Tie2 agonistic antibody rescued the POAG phenotype in Angpt1/Angpt2-deficient mice and rejuvenated the SC in aged mice. These results indicate that the Angpt-Tie2 system is essential for SC integrity. The impairment of this system underlies POAG-associated pathogenesis, supporting the possibility that Tie2 agonists could be a therapeutic option for glaucoma.
Project description:In glaucoma, aqueous outflow into the Schlemm's canal (SC) is obstructed. Despite striking structural and functional similarities with the lymphatic vascular system, it is unknown whether the SC is a blood or lymphatic vessel. Here, we demonstrated the expression of lymphatic endothelial cell markers by the SC in murine and zebrafish models as well as in human eye tissue. The initial stages of SC development involved induction of the transcription factor PROX1 and the lymphangiogenic receptor tyrosine kinase VEGFR-3 in venous endothelial cells in postnatal mice. Using gene deletion and function-blocking antibodies in mice, we determined that the lymphangiogenic growth factor VEGF-C and its receptor, VEGFR-3, are essential for SC development. Delivery of VEGF-C into the adult eye resulted in sprouting, proliferation, and growth of SC endothelial cells, whereas VEGF-A obliterated the aqueous outflow system. Furthermore, a single injection of recombinant VEGF-C induced SC growth and was associated with trend toward a sustained decrease in intraocular pressure in adult mice. These results reveal the evolutionary conservation of the lymphatic-like phenotype of the SC, implicate VEGF-C and VEGFR-3 as critical regulators of SC lymphangiogenesis, and provide a basis for further studies on therapeutic manipulation of the SC with VEGF-C in glaucoma treatment.
Project description:Schlemm's canal (SC) plays central roles in ocular physiology. These roles depend on the molecular phenotypes of SC endothelial cells (SECs). Both the specific phenotype of SECs and development of SC remain poorly defined. To allow a modern and extensive analysis of SC and its origins, we developed a new whole-mount procedure to visualize its development in the context of surrounding tissues. We then applied genetic lineage tracing, specific-fluorescent reporter genes, immunofluorescence, high-resolution confocal microscopy, and three-dimensional (3D) rendering to study SC. Using these techniques, we show that SECs have a unique phenotype that is a blend of both blood and lymphatic endothelial cell phenotypes. By analyzing whole mounts of postnatal mouse eyes progressively to adulthood, we show that SC develops from blood vessels through a newly discovered process that we name "canalogenesis." Functional inhibition of KDR (VEGFR2), a critical receptor in initiating angiogenesis, shows that this receptor is required during canalogenesis. Unlike angiogenesis and similar to stages of vasculogenesis, during canalogenesis tip cells divide and form branched chains prior to vessel formation. Differing from both angiogenesis and vasculogenesis, during canalogenesis SECs express Prox1, a master regulator of lymphangiogenesis and lymphatic phenotypes. Thus, SC development resembles a blend of vascular developmental programs. These advances define SC as a unique vessel with a combination of blood vascular and lymphatic phenotypes. They are important for dissecting its functions that are essential for ocular health and normal vision.
Project description:Ocular hypertension in glaucoma develops due to age-related cellular dysfunction in the conventional outflow tract, resulting in increased resistance to aqueous humor outflow. Two cell types, trabecular meshwork (TM) and Schlemm's canal (SC) endothelia, interact in the juxtacanalicular tissue (JCT) region of the conventional outflow tract to regulate outflow resistance. Unlike endothelial cells lining the systemic vasculature, endothelial cells lining the inner wall of SC support a transcellular pressure gradient in the basal to apical direction, thus acting to push the cells off their basal lamina. The resulting biomechanical strain in SC cells is quite large and is likely to be an important determinant of endothelial barrier function, outflow resistance and intraocular pressure. This review summarizes recent work demonstrating how biomechanical properties of SC cells impact glaucoma. SC cells are highly contractile, and such contraction greatly increases cell stiffness. Elevated cell stiffness in glaucoma may reduce the strain experienced by SC cells, decrease the propensity of SC cells to form pores, and thus impair the egress of aqueous humor from the eye. Furthermore, SC cells are sensitive to the stiffness of their local mechanical microenvironment, altering their own cell stiffness and modulating gene expression in response. Significantly, glaucomatous SC cells appear to be hyper-responsive to substrate stiffness. Thus, evidence suggests that targeting the material properties of SC cells will have therapeutic benefits for lowering intraocular pressure in glaucoma.
Project description:Lymphatic malformations (LMs) are vascular anomalies thought to arise from dysregulated lymphangiogenesis. These lesions impose a significant burden of disease on affected individuals. LM pathobiology is poorly understood, hindering the development of effective treatments. In the present studies, immunostaining of LM tissues revealed that endothelial cells lining aberrant lymphatic vessels and cells in the surrounding stroma expressed the stem cell marker, CD133, and the lymphatic endothelial protein, podoplanin. Isolated patient-derived CD133+ LM cells expressed stem cell genes (NANOG, Oct4), circulating endothelial cell precursor proteins (CD90, CD146, c-Kit, VEGFR-2), and lymphatic endothelial proteins (podoplanin, VEGFR-3). Consistent with a progenitor cell identity, CD133+ LM cells were multipotent and could be differentiated into fat, bone, smooth muscle, and lymphatic endothelial cells in vitro. CD133+ cells were compared to CD133- cells isolated from LM fluids. CD133- LM cells had lower expression of stem cell genes, but expressed circulating endothelial precursor proteins and high levels of lymphatic endothelial proteins, VE-cadherin, CD31, podoplanin, VEGFR-3 and Prox1. CD133- LM cells were not multipotent, consistent with a differentiated lymphatic endothelial cell phenotype. In a mouse xenograft model, CD133+ LM cells differentiated into lymphatic endothelial cells that formed irregularly dilated lymphatic channels, phenocopying human LMs. In vivo, CD133+ LM cells acquired expression of differentiated lymphatic endothelial cell proteins, podoplanin, LYVE1, Prox1, and VEGFR-3, comparable to expression found in LM patient tissues. Taken together, these data identify a novel LM progenitor cell population that differentiates to form the abnormal lymphatic structures characteristic of these lesions, recapitulating the human LM phenotype. This LM progenitor cell population may contribute to the clinically refractory behavior of LMs.
Project description:Glaucoma is a leading cause of blindness, afflicting more than 60 million people worldwide. Increased intraocular pressure (IOP) due to impaired aqueous humor drainage is a major risk factor for the development of glaucoma. Here, we demonstrated that genetic disruption of the angiopoietin/TIE2 (ANGPT/TIE2) signaling pathway results in high IOP, buphthalmos, and classic features of glaucoma, including retinal ganglion degeneration and vision loss. Eyes from mice with induced deletion of Angpt1 and Angpt2 (A1A2Flox(WB) mice) lacked drainage pathways in the corneal limbus, including Schlemm's canal and lymphatic capillaries, which share expression of the PROX1, VEGFR3, and FOXC family of transcription factors. VEGFR3 and FOXCs have been linked to lymphatic disorders in patients, and FOXC1 has been linked to glaucoma. In contrast to blood endothelium, in which ANGPT2 is an antagonist of ANGPT1, we have shown that both ligands cooperate to regulate TIE2 in the lymphatic network of the eye. While A1A2Flox(WB) mice developed high IOP and glaucoma, expression of ANGPT1 or ANGPT2 alone was sufficient for ocular drainage. Furthermore, we demonstrated that loss of FOXC2 from lymphatics results in TIE2 downregulation, suggesting a mechanism for ocular defects in patients with FOXC mutations. These data reveal a pathogenetic and molecular basis for glaucoma and demonstrate the importance of angiopoietin ligand cooperation in the lymphatic endothelium.
Project description:The lymphatic vasculature preserves tissue fluid balance by absorbing fluid and macromolecules and transporting them to the blood vessels for circulation. The stepwise process leading to the formation of the mammalian lymphatic vasculature starts by the expression of the gene Prox1 in a subpopulation of blood endothelial cells (BECs) on the cardinal vein (CV) at approximately E9.5. These Prox1-expressing lymphatic endothelial cells (LECs) will exit the CV to form lymph sacs, primitive structures from which the entire lymphatic network is derived. Until now, no conclusive information was available regarding the cellular processes by which these LEC progenitors exit the CV without compromising the vein's integrity. We determined that LECs leave the CV by an active budding mechanism. During this process, LEC progenitors are interconnected by VE-cadherin-expressing junctions. Surprisingly, we also found that Prox1-expressing LEC progenitors were present not only in the CV but also in the intersomitic vessels (ISVs). Furthermore, as LEC progenitors bud from the CV and ISVs into the surrounding mesenchyme, they begin expressing the lymphatic marker podoplanin, migrate away from the CV, and form the lymph sacs. Analyzing this process in Prox1-null embryos revealed that Prox1 activity is necessary for LEC progenitors to exit the CV.
Project description:In the past decade, many new pharmacological and surgical treatments have become available to lower intraocular pressure (IOP) for glaucoma. The majority of these options have targeted improving aqueous humor outflow (AHO). At the same time, in addition to new treatments, research advances in AHO assessment have led to the development of new tools to structurally assess AHO pathways and to visualize where aqueous is flowing in the eye. These new imaging modalities have uncovered novel AHO observations that challenge traditional AHO concepts. New behaviors including segmental, pulsatile, and dynamic AHO may have relevance to the disease and the level of therapeutic response for IOP-lowering treatments. By better understanding the regulation of segmental, pulsatile, and dynamic AHO, it may be possible to find new and innovative treatments for glaucoma aiming at these new AHO behaviors.
Project description:In development, lymphatic endothelial cells originate within veins and differentiate via a process requiring Prox1. Notch signaling regulates cell-fate decisions, and expression studies suggested that Jag1/Notch1 signaling functions in veins during lymphatic endothelial specification. Using an inducible lymphatic endothelial Prox1CreER(T2) driver, Notch signaling was suppressed by deleting Notch1 or expressing dominant-negative Mastermind-like in Prox1+ endothelial cells. Either loss of Notch1 or reduced Notch signaling increased Prox1+ lymphatic endothelial progenitor cell numbers in the veins, leading to incomplete separation of venous and lymphatic vessels. Notch loss of function resulted in excessive Prox1+ lymphatic cells emerging from the cardinal vein and significant lymphatic overgrowth. Moreover, loss of one allele of Notch1 in Prox1 heterozygous mice rescued embryonic lethality due to Prox1 haploinsufficiency and significantly increased Prox1+ lymphatic endothelial progenitor cell numbers. Expression of a constitutively active Notch1 protein in Prox1+ cells suppressed endothelial Prox1 from E9.75 to E13.5, resulting in misspecified lymphatic endothelial cells based upon reduced expression of podoplanin, LYVE1 and VEGFR3. Notch activation resulted in the appearance of blood endothelial cells in peripheral lymphatic vessels. Activation of Notch signaling in the venous endothelium at E10.5 did not arterialize the cardinal vein, suggesting that Notch can no longer promote arterialization in the cardinal vein during this developmental stage. We report a novel role for Notch1 in limiting the number of lymphatic endothelial cells that differentiate from the veins to assure proper lymphatic specification.
Project description:Aqueous humor drainage is essential for the regulation of intraocular pressure (IOP), a major risk factor for glaucoma. The Schlemm’s canal and the non-conventional uveoscleral pathway are known to drain aqueous humor from the eye anterior chamber. It has recently been reported that lymphatic vessels are involved in this process, and that the Schlemm’s canal responds to some lymphatic regulators. We have previously shown a critical role for bone morphogenetic protein 9 (BMP9) in lymphatic vessel maturation and valve formation, with repercussions in drainage efficiency. Here, we imaged eye lymphatic vessels and analyzed the consequences of Bmp9 (Gdf2) gene invalidation. A network of lymphatic vessel hyaluronan receptor 1 (LYVE-1)-positive lymphatic vessels was observed in the corneolimbus and the conjunctiva. In contrast, LYVE-1-positive cells present in the ciliary bodies were belonging to the macrophage lineage. Although enlarged conjunctival lymphatic trunks and a reduced valve number were observed in Bmp9-KO mice, there were no morphological differences in the Schlemm’s canal compared to wild type animals. Moreover, there were no functional consequences on IOP in both basal control conditions and after laser-induced ocular hypertonia. Thus, the BMP9-activated signaling pathway does not constitute a wise target for new glaucoma therapeutic strategies.