Sunju Lee

Visualization of lymphatic vessels by Prox1-promoter directed GFP reporter in a bacterial artificial chromosome-based transgenic mouse

Although the blood vessel-specific fluorescent transgenic mouse has been an excellent tool to study vasculogenesis and angiogenesis, a lymphatic-specific fluorescent mouse model has not been established to date. Here we report a transgenic animal model that expresses the green fluorescent protein under the promoter of Prox1, a master control gene in lymphatic development. Generated using an approximately 200-kb-long bacterial artificial chromosome harboring the entire Prox1 gene, this Prox1-green fluorescent protein mouse was found to faithfully recapitulate the expression pattern of the Prox1 gene in lymphatic endothelial cells and other Prox1-expressing organs, and enabled us to conveniently visualize detailed structure and morphology of lymphatic vessels and networks throughout development. Our data demonstrate that this novel transgenic mouse can be extremely useful for detection, imaging, and isolation of lymphatic vessels and monitoring wound-associated lymphangiogenesis. Together, this Prox1-green fluorescent protein transgenic mouse will be a great tool for the lymphatic research.

Prox1 physically and functionally interacts with COUP-TFII to specify lymphatic endothelial cell fate

Specification of endothelial cell (EC) fate during vascular development is controlled by distinct key regulators. While Notch plays an essential role in induction of arterial phenotypes, COUP-TFII is required to maintain the venous EC identity. Homeodomain transcription factor Prox1 functions to reprogram venous ECs to lymphatic endothelial cells (LECs). Here, we report that the venous EC fate regulator COUP-TFII is expressed in LECs throughout development and physically interacts with Prox1 to form a stable complex in various cell types including LECs. We found that COUP-TFII functions as a coregulator of Prox1 to control several lineage-specific genes including VEGFR-3, FGFR-3, and neuropilin-1 and is required along with Prox1 to maintain LEC phenotype. Together, we propose that the physical and functional interactions of the 2 proteins constitute an essential part in the program specifying LEC fate and may provide the molecular basis for the hypothesis of venous EC identity being the prerequisite for LEC specification.

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.

Lymphatic regulator PROX1 determines Schlemm’s canal integrity and identity

Schlemms 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 α9 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.

The New Era of the Lymphatic System: No Longer Secondary to the Blood Vascular System

The blood and lymphatic systems are the two major circulatory systems in our body. Although the blood system has been studied extensively, the lymphatic system has received much less scientific and medical attention because of its elusive morphology and mysterious pathophysiology. However, a series of landmark discoveries made in the past decade has begun to change the previous misconception of the lymphatic system to be secondary to the more essential blood vascular system. In this article, we review the current understanding of the development and pathology of the lymphatic system. We hope to convince readers that the lymphatic system is no less essential than the blood circulatory system for human health and well-being.

9-Cis Retinoic Acid Promotes Lymphangiogenesis and Enhances Lymphatic Vessel Regeneration: Therapeutic Implications of 9-Cis Retinoic Acid for Secondary Lymphedema

Background— The lymphatic system plays a key role in tissue fluid homeostasis and lymphatic dysfunction caused by genetic defects, or lymphatic vessel obstruction can cause lymphedema, disfiguring tissue swelling often associated with fibrosis and recurrent infections with no available cures to date. In this study, retinoic acids (RAs) were determined to be a potent therapeutic agent that is immediately applicable to reduce secondary lymphedema. Methods and Results— We report that RAs promote proliferation, migration, and tube formation of cultured lymphatic endothelial cells by activating fibroblast growth factor receptor signaling. Moreover, RAs control the expression of cell-cycle checkpoint regulators such as p27Kip1, p57Kip2, and the aurora kinases through both an Akt-mediated nongenomic action and a transcription-dependent genomic action that is mediated by Prox1, a master regulator of lymphatic development. Moreover, 9-cisRA was found to activate in vivo lymphangiogenesis in animals in mouse trachea, Matrigel plug, and cornea pocket assays. Finally, we demonstrate that 9-cisRA can provide a strong therapeutic efficacy in ameliorating experimental mouse tail lymphedema by enhancing lymphatic vessel regeneration. Conclusion— These in vitro and animal studies demonstrate that 9-cisRA potently activates lymphangiogenesis and promotes lymphatic regeneration in an experimental lymphedema model, presenting it as a promising novel therapeutic agent to treat human lymphedema patients. # Clinical Perspective {#article-title-36}Background— The lymphatic system plays a key role in tissue fluid homeostasis and lymphatic dysfunction caused by genetic defects, or lymphatic vessel obstruction can cause lymphedema, disfiguring tissue swelling often associated with fibrosis and recurrent infections with no available cures to date. In this study, retinoic acids (RAs) were determined to be a potent therapeutic agent that is immediately applicable to reduce secondary lymphedema. Methods and Results— We report that RAs promote proliferation, migration, and tube formation of cultured lymphatic endothelial cells by activating fibroblast growth factor receptor signaling. Moreover, RAs control the expression of cell-cycle checkpoint regulators such as p27Kip1, p57Kip2, and the aurora kinases through both an Akt-mediated nongenomic action and a transcription-dependent genomic action that is mediated by Prox1, a master regulator of lymphatic development. Moreover, 9-cisRA was found to activate in vivo lymphangiogenesis in animals in mouse trachea, Matrigel plug, and cornea pocket assays. Finally, we demonstrate that 9-cisRA can provide a strong therapeutic efficacy in ameliorating experimental mouse tail lymphedema by enhancing lymphatic vessel regeneration. Conclusion— These in vitro and animal studies demonstrate that 9-cisRA potently activates lymphangiogenesis and promotes lymphatic regeneration in an experimental lymphedema model, presenting it as a promising novel therapeutic agent to treat human lymphedema patients.

Kaposin-B enhances the PROX1 mRNA stability during lymphatic reprogramming of vascular endothelial cells by Kaposi's sarcoma herpes virus.

Kaposis sarcoma (KS) is the most common cancer among HIV-positive patients. Histogenetic origin of KS has long been elusive due to a mixed expression of both blood and lymphatic endothelial markers in KS tumor cells. However, we and others discovered that Kaposis sarcoma herpes virus (KSHV) induces lymphatic reprogramming of blood vascular endothelial cells by upregulating PROX1, which functions as the master regulator for lymphatic endothelial differentiation. Here, we demonstrate that the KSHV latent gene kaposin-B enhances the PROX1 mRNA stability and plays an important role in KSHV-mediated PROX1 upregulation. We found that PROX1 mRNA contains a canonical AU-rich element (ARE) in its 3′-untranslated region that promotes PROX1 mRNA turnover and that kaposin-B stimulates cytoplasmic accumulation of the ARE-binding protein HuR through activation of the p38/MK2 pathway. Moreover, HuR binds to and stabilizes PROX1 mRNA through its ARE and is necessary for KSHV-mediated PROX1 mRNA stabilization. Together, our study demonstrates that kaposin-B plays a key role in PROX1 upregulation during lymphatic reprogramming of blood vascular endothelial cells by KSHV.

Automated characterization of pigment epithelial detachment by optical coherence tomography.

PURPOSE To assess the accuracy of automated classification of pigment epithelial detachments (PED) by using a software algorithm applied to spectral-domain optical coherence tomography (SD-OCT) scans. METHODS HD-OCT (Cirrus; Carl Zeiss Meditec, Dublin, CA) volume scans (512 × 128) were retrospectively collected from 46 eyes of 33 patients with evidence of PED in the setting of age-related macular degeneration (AMD, n = 28) or central serous chorioretinopathy (CSCR, n = 5). In these eyes, 168 PEDs were automatically detected with a system-associated tool (Cirrus HD-OCT RPE Elevation Analysis; Carl Zeiss Meditec). Two independent, certified Doheny Image Reading Center (DIRC) OCT graders classified these PEDs into three categories--serous, drusenoid, or fibrovascular--via inspection of the B-scans. Manual classification results served as the gold standard for comparisons with automated classification. For automated classification, interindividual variation in intensities was normalized in all images. Individual A-scans within the detected PEDs were then automatically classified into one of three categories based on the mean internal intensity and the standard deviation of the internal intensity: mean intensity <30 (serous type); mean intensity ≥30 but <60 or mean intensity ≥30 and SD ≥30 (fibrovascular type); or mean intensity ≥60 and SD < 30 (drusenoid type). Individual PEDs were then automatically classified into the same three categories based on the predominant type of A-scan within the PED. For mixed PEDs (many A-scans of each type), a risk index for neovascularization was computed based on the percentage of fibrovascular A-scans. In addition, a confidence index was computed for each PED based on its mathematical distance from the PED category boundaries. RESULTS Among the 168 PEDs, the DIRC graders classified 16 as serous, 88 as fibrovascular, and 64 as drusenoid PEDs. The automated algorithm classified 14 as serous, 96 as fibrovascular, and 58 as drusenoid PEDs. The sensitivity and specificity values for automated classification according to type of PED were 88% and 100% for serous, 76% and 64% for fibrovascular, and 58% and 81% for drusenoid, respectively. CONCLUSIONS Automated classification of PEDs using internal reflectivity characteristics appears to be sensitive for detecting serous and fibrovascular PEDs. Automated classification and quantification of PEDs may be a useful tool in future studies for stratifying PEDs according to risk and possibly predicting the risk of advanced AMD.

Interleukin-8 reduces post-surgical lymphedema formation by promoting lymphatic vessel regeneration

Lymphedema is mainly caused by lymphatic obstruction and manifested as tissue swelling, often in the arms and legs. Lymphedema is one of the most common post-surgical complications in breast cancer patients and presents a painful and disfiguring chronic illness that has few treatment options. Here, we evaluated the therapeutic potential of interleukin (IL)-8 in lymphatic regeneration independent of its pro-inflammatory activity. We found that IL-8 promoted proliferation, tube formation, and migration of lymphatic endothelial cells (LECs) without activating the VEGF signaling. Additionally, IL-8 suppressed the major cell cycle inhibitor CDKN1C/p57KIP2 by downregulating its positive regulator PROX1, which is known as the master regulator of LEC-differentiation. Animal-based studies such as matrigel plug and cornea micropocket assays demonstrated potent efficacy of IL-8 in activating lymphangiogenesis in vivo. Moreover, we have generated a novel transgenic mouse model (K14-hIL8) that expresses human IL-8 in the skin and then crossed with lymphatic-specific fluorescent (Prox1-GFP) mouse. The resulting double transgenic mice showed that a stable expression of IL-8 could promote embryonic lymphangiogenesis. Moreover, an immunodeficient IL-8-expressing mouse line that was established by crossing K14-hIL8 mice with athymic nude mice displayed an enhanced tumor-associated lymphangiogenesis. Finally, when experimental lymphedema was introduced, K14-hIL8 mice showed an improved amelioration of lymphedema with an increased lymphatic regeneration. Together, we report that IL-8 can activate lymphangiogenesis in vitro and in vivo with a therapeutic efficacy in post-surgical lymphedema.

Heterogeneity and plasticity of lymphatic endothelial cells.

Endothelial cells are found in most organs and tissues in our body. Despite their apparent morphological and functional similarities, endothelial cells exhibit remarkable heterogeneity and plasticity. In a strict sense, no two endothelial cells are identical in terms of their biological, immunological, functional, metabolic, morphological, and anatomical aspects. Their heterogeneity and plasticity are now known to be dependent upon and conferred by their microenvironments, arteriovenous-lymphatic cell identity, organ-specific vascular beds, fluid dynamics, vessel sizes, anatomical locations, physiological and pathological states, and more. Although abundant evidence is available to demonstrate endothelial heterogeneity in the blood vascular system, studies of heterogeneity and plasticity of lymphatic endothelial cells are limited because of the short history of lymphatic research. Nonetheless, a growing body of exciting work has begun to discover that lymphatic endothelial cells are as heterogeneous as blood vascular endothelial cells. In this article, we discuss the heterogeneity and plasticity of lymphatic endothelial cells.