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Background and Aims: Secretion and transport of intestinal chylomicrons (CMs) via lymphatics to the blood circulation is stimulated primarily by fat ingestion, whereas several other factors have also been shown to play important roles in regulating CM secretion rate. Among these factors, active regulation of lymphatic pumping has not been appreciated to date. The gut peptide and intestinal growth factor glucagon-like peptide-2 (GLP-2) has emerged as a robust enhancer of intestinal lipid mobilization and secretion. The present study aims to elucidate GLP-2’s impact on lacteal contractility and assess enteric nervous system (ENS) involvement in GLP-2–induced effects on lipid mobilization. Methods: Using intravital imaging of a prospero-related homeobox 1-enhanced green fluorescent protein rat model, we assessed GLP-2’s effect on lacteal contractility, in the presence and absence of the ENS inhibitor mecamylamine (MEC). Concurrently, to explore the physiological relevance, we examined GLP-2’s impact on lymph flow and triglyceride (TG) output in vivo in a rat lymph fistula model. Results: GLP-2 significantly increased lacteal contractility, and this effect was inhibited by MEC. In the rat lymph fistula model, GLP-2 increased lymph flow, lymph volume, cumulative lymph volume, and TG output while reducing lymph TG concentration. MEC administration blocked these effects of GLP-2. Peak enhancement of lacteal contractility and enhancement of lymph flow in vivo occurred simultaneously with maximal effect at 15–20 minutes post GLP-2 administration, suggesting that GLP-2 enhances lipid transport by stimulating lymphatic contractility. Conclusion: For the first time, through imaging and concurrent rat lymphatic fistula studies, we demonstrated active regulation of lymphatic contractility as a key determinant of CM secretion and that intact ENS was required to observe this effect.

Abstract Primary open angle glaucoma (POAG) features an optic neuropathy, elevated aqueous humor (AH) TGFβ2, and major risk factors of central corneal thickness (CCT), increasing age and intraocular pressure (IOP). We examined Tight skin (Tsk) mice to see if mutation of fibrillin-1, a repository for latent TGFβ, is associated with characteristics of human POAG. We measured: CCT by ocular coherence tomography (OCT); IOP; retinal ganglion cell (RGC) and optic nerve axon counts by microscopic techniques; visual electrophysiologic scotopic threshold responses (STR) and pattern electroretinogram (PERG); and AH TGFβ2 levels and activity by ELISA and MINK epithelial cell-based assays respectively. Tsk mice had open anterior chamber angles and compared with age-matched wild type (WT) mice: 23% thinner CCT (p

Abstract Background In Japan, six workers handling cross-linked water-soluble acrylic acid polymer (CWAAP) at a chemical plant suffered from lung diseases, including fibrosis, interstitial pneumonia, emphysema, and pneumothorax. We recently demonstrated that inhalation of CWAAP-A, one type of CWAAP, causes pulmonary disorders in rats. It is important to investigate dose–response relationships and recoverability from exposure to CWAAPs for establishing occupational health guidelines, such as setting threshold limit value for CWAAPs in the workplace. Methods Male and female F344 rats were exposed to 0.3, 1, 3, or 10 mg/m3 CWAAP-A for 6 h/day, 5 days/week for 13 weeks using a whole-body inhalation exposure system. At 1 h, 4 weeks, and 13 weeks after the last exposure the rats were euthanized and blood, bronchoalveolar lavage fluid, and all tissues including lungs and mediastinal lymph nodes were collected and subjected to biological and histopathological analyses. In a second experiment, male rats were pre-treated with clodronate liposome or polymorphonuclear leukocyte-neutralizing antibody to deplete macrophages or neutrophils, respectively, and exposed to CWAAP-A for 6 h/day for 2 days. Results CWAAP-A exposure damaged only the alveoli. The lowest observed adverse effect concentration (LOAEC) was 1 mg/m3 and the no observed adverse effect concentration (NOAEC) was 0.3 mg/m3. Rats of both sexes were able to recover from the tissue damage caused by 13 weeks exposure to 1 mg/m3 CWAAP-A. In contrast, tissue damage caused by exposure to 3 and 10 mg/m3 was irreversible due to the development of interstitial lung lesions. There was a gender difference in the recovery from CWAAP-A induced pulmonary disorders, with females recovering less than males. Finally, acute lung effects caused by CWAAP-A were significantly reduced by depletion of alveolar macrophages. Conclusions Pulmonary damage caused by inhalation exposure to CWAAP-A was dose-dependent, specific to the lung and lymph nodes, and acute lung damage was ameliorated by depleting macrophages in the lungs. CWAAP-A had both a LOAEC and a NOAEC, and tissue damage caused by exposure to 1 mg/m3 CWAAP-A was reversible: recovery in female rats was less than for males. These findings indicate that concentration limits for CWAAPs in the workplace can be determined.

Abstract Microfluidic organ-on-a-chip technologies have enabled construction of biomimetic physiologically and pathologically relevant models. This paper describes an injection molded microfluidic platform that utilizes a novel sequential edge-guided patterning method based on spontaneous capillary flow to realize three-dimensional co-culture models and form an array of micro-vascularized tissues (28 per 1 × 2-inch slide format). The MicroVascular Injection-Molded Plastic Array 3D Culture (MV-IMPACT) platform is fabricated by injection molding, resulting in devices that are reliable and easy to use. By patterning hydrogels containing human umbilical endothelial cells and fibroblasts in close proximity and allowing them to form vasculogenic networks, an array of perfusable vascularized micro-tissues can be formed in a highly efficient manner. The high-throughput generation of angiogenic sprouts was quantified and their uniformity was characterized. Due to its compact design (half the size of a 96-well microtiter plate), it requires small amount of reagents and cells per device. In addition, the device design is compatible with a high content imaging machine such as Yokogawa CQ-1. Furthermore, we demonstrated the potential of our platform for high-throughput phenotypic screening by testing the effect of DAPT, a chemical known to affect angiogenesis. The MV-IMPACT represent a significant improvement over our previous PDMS-based devices in terms of molding 3D co-culture conditions at much higher throughput with added reliability and robustness in obtaining vascular micro-tissues and will provide a platform for developing applications in drug screening and development.

Research on the development of anti-cancer drugs has progressed, but the low reliability of animal experiments due to biological differences between animals and humans causes failures in the clinical process. To overcome this limitation, 3-dimensional (3D) in vitro models have been developed to mimic the human cellular microenvironment using polydimethylsiloxane (PDMS). However, due to the characteristics and limitations of PDMS, it has low efficiency and is not suitable to be applied in the preclinical testing of a drug. High-throughput microfluidic platforms fabricated by injection molding have been developed, but these platforms require a laborious process when handling spheroids. We recently developed an injection-molded plastic array 3D culture tissue platform that integrates the process from spheroid formation to reconstruction of an in vitro model with spheroids (All-in-One-IMPACT). In this study, we implemented a 3D tumor spheroid angiogenesis model in the developed platform. We analyzed the tendency for angiogenesis according to gel concentration and confirmed that angiogenesis occurred using cancer cell lines and patient-derived cancer cells (PDCs). We also administered an anti-cancer drug to the PDC tumor spheroid angiogenesis model to observe the drug’s effect on angiogenesis according to its concentration. We demonstrated that our platform can be used to study the tumor microenvironment (TME) and drug screening. We expect that this platform will contribute to further research on the complex mechanisms of the TME and predictive preclinical models.

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Purpose: To uncover the mechanism of subconjunctival outflow in humans. Design: Cross-sectional study. Participants: Fifteen patients receiving subconjunctival anesthesia before intravitreal injection for routine clinical care. Methods: Anterior segment (AS) OCT was performed in patients with various instances of conjunctival edema or subconjunctival fluid. Other patients received a subconjunctival mixture of 0.005% indocyanine green and 2% lidocaine. After subconjunctival injection of the tracer and anesthetic mixture, blebs and associated outflow pathways were imaged angiographically and the time for appearance was recorded. The pattern and structure of outflow pathways were studied using AS OCT. Angiographic and AS OCT results were compared with trabecular and conventional outflow imaging, which demonstrates veins. Main Outcome Measures: Ocular surface lymphangiography and AS OCT images. Results: Anterior segment OCT of the conjunctiva in a normal eye demonstrated thin nonedematous conjunctiva with absent intraconjunctival lumens or subconjunctival fluid. Patients with a history of trabeculectomy, subconjunctival drug injection, or chemosis demonstrated thickened conjunctiva and intraconjunctival luminal pathways that contained valve-like structures. Tracer-based studies in patients demonstrated blebs with irregular subconjunctival bleb-related outflow patterns that arose in a time-dependent fashion. These angiographic pathways were luminal on OCT, sausage shaped, and contained intraluminal valve-like structures. This was in contrast to trabecular and conventional outflow imaging, where pathways were classically Y-shaped, of even caliber, and lacked valve-like structures. Conclusions: Outflow pathways were seen in patients with conjunctival edema and after subconjunctival tracer injection. These pathways were lymphatic based on pattern and structural study. Better understanding of bleb-related lymphatic outflow may lead to improved bleb-requiring glaucoma surgeries and subconjunctival drug delivery.

Abstract Ionizing radiation, commonly used in the treatment of solid tumors, has unintended but deleterious effects on overlying skin and is associated with chronic nonhealing wounds. Skin‐derived mesenchymal stromal cells (SMSCs) are a pluripotent population of cells that are critically involved in skin homeostasis and wound healing. The aim of this study was to isolate and functionally characterize SMSCs from human skin that was previously irradiated as part of neoadjuvant or adjuvant cancer therapy. To this end, SMSCs were isolated from paired irradiated and nonirradiated human skin samples. Irradiated SMSCs expressed characteristic SMSC markers at lower levels, had disorganized cytoskeletal structure, and had disordered morphology. Functionally, these cells had diminished proliferative capacity and substantial defects in colony‐forming capacity and differentiation in vitro. These changes were associated with significant differential expression of genes known to be involved in skin physiology and wound healing. Conditioned media obtained from irradiated SMSCs affected fibroblast but not endothelial cell proliferation and migration. These results suggest that in situ damage to SMSCs during neoadjuvant or adjuvant radiation may play a critical role in the pathogenesis of slow or nonhealing radiation wounds. Stem Cells Translational Medicine 2019;8:925&934

Endothelial cells are important in the maintenance of healthy blood vessels and in the development of vascular diseases. However, the origin and dynamics of endothelial precursors and remodeling at the single-cell level have been difficult to study in vivo owing to technical limitations. Therefore, we aimed to develop a direct visual approach to track the fate and function of single endothelial cells over several days and weeks in the same vascular bed in vivo using multiphoton microscopy (MPM) of transgenic Cdh5-Confetti mice and the kidney glomerulus as a model. Individual cells of the vascular endothelial lineage were identified and tracked owing to their unique color combination, based on the random expression of cyan/green/yellow/red fluorescent proteins. Experimental hypertension, hyperglycemia, and laser-induced endothelial cell ablation rapidly increased the number of new glomerular endothelial cells that appeared in clusters of the same color, suggesting clonal cell remodeling by local precursors at the vascular pole. Furthermore, intravital MPM allowed the detection of distinct structural and functional alterations of proliferating endothelial cells. No circulating Cdh5-Confetti+ cells were found in the renal cortex. Moreover, the heart, lung, and kidneys showed more significant clonal endothelial cell expansion compared with the brain, pancreas, liver, and spleen. In summary, we have demonstrated that serial MPM of Cdh5-Confetti mice in vivo is a powerful technical advance to study endothelial remodeling and repair in the kidney and other organs under physiological and disease conditions.

VISTA is an up-and-coming immune checkpoint molecule that can become the target of new cancer immunotherapy treatments. Immune cells in the tumor microenvironment can largely influence the progression of cancer through inhibitory and stimulatory pathways. Indeed, VISTA is expressed on many immune cells, including T cells, myeloid-derived suppressor cells, tumor-associated macrophages, and dendritic cells. VISTA has predominantly been shown to act in an immune-suppressing manner that enables cancer progression. This review will delve into results from preclinical murine studies of anti-VISTA monoclonal antibody treatments, bring together recent studies that detect VISTA expression on immune cells from patient tumors of various cancers, and discuss ongoing clinical trials involving VISTA.

Over the past couple of decades, lymphatics research has accelerated and gained a much-needed recognition in pathophysiology. As the lymphatic system plays heavy roles in interstitial fluid drainage, immune surveillance and lipid absorption, the ablation or excessive growth of this vasculature could be associated with many complications, from lymphedema to metastasis. Despite their growing importance in cancer, few anti-lymphangiogenic therapies exist today, as they have yet to pass phase 3 clinical trials and acquire FDA approval. As such, many studies are being done to better define the signaling pathways that govern lymphangiogenesis, in hopes of developing new therapeutic approaches to inhibit or stimulate this process. This review will cover our current understanding of the Ras signaling pathways and their interactions with Prox1, the master transcriptional switch involved in specifying lymphatic endothelial cell fate and lymphangiogenesis, in hopes of providing insights to lymphangiogenesis-based therapies.

The MTOR signal is known to be activated in various cancer cells including hepatocellular carcinoma (HCC) cells. Rapamycin, a specific inhibitor of MTOR, has been widely used as an immunosuppressant in organ transplant patients, and its clinical application has been recently expanded to cancer therapy. In this study, the anti-proliferative effect of rapamycin was investigated in four different HCC cell lines. Rapamycin effectively inhibited the proliferation of Huh7 or Hep3B, but not that of HepG2 or SNU3160 cells. Interestingly, rapamycin increased Prospero-related homeobox 1 (PROX1) expression at the protein level, but did not affect its transcript in Huh7 as well as Hep3B cells. Moreover, immunoprecipitation assays showed that PROX1 ubiquitination was downregulated by rapamycin. Furthermore, PROX1 over-expression or siRNA knock-down in Huh7 and Hep3B cells reduced or increased proliferation, respectively. The effect of PROX1 over-expression on the sensitivity to rapamycin was not synergistic, but the effect of MTOR inhibition on cell proliferation was diminished by PROX1 siRNA. Finally, Huh7 cells were inoculated into the flanks of nude mice and rapamycin was injected daily for 14 days. The xenograft volume was decreased and PROX1 expression was increased by rapamycin. These results indicate that PROX1 plays a key role in the anti-proliferative effect of rapamycin and suggest that the increased PROX1 by MTOR inhibition can be used as a useful marker for predicting whether HCC cells can be affected by rapamycin.

Abstract Outflow resistance in the aqueous drainage tract distal to trabecular meshwork is potentially an important determinant of intraocular pressure and success of trabecular bypass glaucoma surgeries. It is unclear how distal resistance is modulated. We sought to establish: (a) multimodal 2-photon deep tissue imaging and 3-dimensional analysis of the distal aqueous drainage tract (DT) in transgenic mice in vivo and ex vivo; (b) criteria for distinguishing the DT from blood and lymphatic vessels; and (c) presence of a DT wall organization capable of contractility. DT lumen appeared as scleral collagen second harmonic generation signal voids that could be traced back to Schlemm’s canal. DT endothelium was Prox1-positive, CD31-positive and LYVE-1-negative, bearing a different molecular signature from blood and true lymphatic vessels. DT walls showed prominent filamentous actin (F-actin) labeling reflecting cells in a contracted state. F-actin co-localized with mesenchymal smooth muscle epitopes of alpha-smooth muscle actin, caldesmon and calponin, which localized adjacent and external to the endothelium. Our findings support a DT wall organization resembling that of blood vessels. This reflects a capacity to contract and support dynamic alteration of DT caliber and resistance analogous to the role of blood vessel tone in regulating blood flow.

Abstract The lymphatic system plays a key role in tissue fluid homeostasis, immune cell trafficking, and fat absorption. We previously reported a bacterial artificial chromosome (BAC)-based lymphatic reporter mouse, where EGFP is expressed under the regulation of the Prox1 promoter. This reporter line has been widely used to conveniently visualize lymphatic vessels and other Prox1-expressing tissues such as Schlemm’s canal. However, mice have a number of experimental limitations due to small body size. By comparison, laboratory rats are larger in size and more closely model the metabolic, physiological, and surgical aspects of humans. Here, we report development of a novel lymphatic reporter rat using the mouse Prox1-EGFP BAC. Despite the species mismatch, the mouse Prox1-EGFP BAC enabled a reliable expression of EGFP in Prox1-expressing cells of the transgenic rats and allowed a convenient visualization of all lymphatic vessels, including those in the central nervous system, and Schlemm’s canal. To demonstrate the utility of this new reporter rat, we studied the contractile properties and valvular functions of mesenteric lymphatics, developed a surgical model for vascularized lymph node transplantation, and confirmed Prox1 expression in venous valves. Together, Prox1-EGFP rat model will contribute to the advancement of lymphatic research as a valuable experimental resource.

Abstract Wound healing is significantly delayed in irradiated skin. To better understand global changes in protein expression after radiation, we utilized a reverse phase protein array (RPPA) to identify significant changes in paired samples of normal and irradiated human skin. Of the 210 proteins studied, fibronectin was the most significantly and consistently downregulated in radiation-damaged skin. Using a murine model, we confirmed that radiation leads to decreased fibronectin expression in the skin as well as delayed wound healing. Topically applied fibronectin was found to significantly improve wound healing in irradiated skin and was associated with decreased inflammatory infiltrate and increased angiogenesis. Fibronectin treatment may be a useful adjunctive modality in the treatment of non-healing radiation wounds.

Vascularized lymph node transfer (VLNT) is a promising treatment modality for lymphedema; however, how lymphatic tissue responds to ischemia has not been well defined. This study investigates the cellular changes that occur in lymph nodes in response to ischemia and reperfusion. Lymph node containing superficial epigastric artery-based groin flaps were isolated in Prox-1 EGFP rats which permits real time identification of lymphatic tissue by green fluorescence during flap dissection. Flaps were subjected to ischemia for either 1, 2, 4, or 8 hours, by temporarily occluding the vascular pedicle. Flaps were harvested after 0 hours, 24 hours, or 5 days of reperfusion. Using EGFP signal guidance, lymph nodes were isolated from the flaps and tissue morphology, cell apoptosis, and inflammatory cytokines were quantified and analyzed via histology, immunostaining, and rtPCR. There was a significant increase in collagen deposition and tissue fibrosis in lymph nodes after 4 and 8 hours of ischemia compared to 1 and 2 hours, as assessed by picrosirius red staining. Cell apoptosis significantly increased after 4 hours of ischemia in all harvest times. In tissue subject to 4 hours of ischemia, longer reperfusion periods were associated with increased rates of CD3+ and CD45+ cell apoptosis. rtPCR analysis demonstrated significantly increased expression of CXCL1/GRO-α with 2 hours of ischemia and increased PECAM-1 and TNF-α expression with 1 hour of ischemia. Significant cell death and changes in tissue morphology do not occur until after 4 hours of ischemia; however, analysis of inflammatory biomarkers suggests that ischemia reperfusion injury can occur with as little as 2 hours of ischemia.

Summary: Wnt/β-catenin signaling is necessary for lymphatic vascular development. Oscillatory shear stress (OSS) enhances Wnt/β-catenin signaling in cultured lymphatic endothelial cells (LECs) to induce expression of the lymphedema-associated transcription factors GATA2 and FOXC2. However, the mechanisms by which OSS regulates Wnt/β-catenin signaling and GATA2 and FOXC2 expression are unknown. We show that OSS activates autocrine Wnt/β-catenin signaling in LECs in vitro. Tissue-specific deletion of Wntless, which is required for the secretion of Wnt ligands, reveals that LECs and vascular smooth muscle cells are complementary sources of Wnt ligands that regulate lymphatic vascular development in vivo. Further, the LEC master transcription factor PROX1 forms a complex with β-catenin and the TCF/LEF transcription factor TCF7L1 to enhance Wnt/β-catenin signaling and promote FOXC2 and GATA2 expression in LECs. Thus, our work defines Wnt sources, reveals that PROX1 directs cell fate by acting as a Wnt signaling component, and dissects the mechanisms of PROX1 and Wnt synergy. : Cha et al. demonstrate that lymphatic vascular development is regulated by Wnt ligands secreted by lymphatic endothelial cells (LECs) and vascular smooth muscle cells. Oscillatory shear stress regulates Wnt secretion from LECs. PROX1 interacts with β-catenin and TCF7L1 to regulate Wnt signaling and promote the expression of FOXC2 and GATA2. Keywords: lymphatic vasculature, lymphovenous valves, lymphatic valves, PROX1, GATA2, FOXC2, Wnt/β-catenin signaling, oscillatory shear stress, Wntless, TCF7L1

ABSTRACT Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiologic agent for Kaposi’s sarcoma (KS), which is one of the most common HIV-associated neoplasms. The endothelium is the thin layer of squamous cells where vascular blood endothelial cells (BECs) line the interior surface of blood vessels and lymphatic endothelial cells (LECs) are in direct contact with lymphatic vessels. The KS lesions contain a prominent compartment of neoplastic spindle morphology cells that are closely related to LECs. Furthermore, while KSHV can infect both LECs and BECs in vitro, its infection activates genetic programming related to lymphatic endothelial cell fate, suggesting that lymphangiogenic pathways are involved in KSHV infection and malignancy. Here, we report for the first time that viral interferon regulatory factor 3 (vIRF3) is readily detected in over 40% of KS lesions and that vIRF3 functions as a proangiogenic factor, inducing hypersprouting formation and abnormal growth in a LEC-specific manner. Mass spectrometry analysis revealed that vIRF3 interacted with histone deacetylase 5 (HDAC5), which is a signal-responsive regulator for vascular homeostasis. This interaction blocked the phosphorylation-dependent cytosolic translocation of HDAC5 and ultimately altered global gene expression in LECs but not in BECs. Consequently, vIRF3 robustly induced spindle morphology and hypersprouting formation of LECs but not BECs. Finally, KSHV infection led to the hypersprouting formation of LECs, whereas infection with a ΔvIRF3 mutant did not do so. Collectively, our data indicate that vIRF3 alters global gene expression and induces a hypersprouting formation in an HDAC5-binding-dependent and LEC-specific manner, ultimately contributing to KSHV-associated pathogenesis. IMPORTANCE Several lines of evidences indicate that KSHV infection of LECs induces pathological lymphangiogenesis and that the results resemble KS-like spindle morphology. However, the underlying molecular mechanism remains unclear. Here, we demonstrated that KSHV vIRF3 is readily detected in over 40% of various KS lesions and functions as a potent prolymphangiogenic factor by blocking the phosphorylation-dependent cytosolic translocation of HDAC5, which in turn modulates global gene expression in LECs. Consequently, vIRF3-HDAC5 interaction contributes to virus-induced lymphangiogenesis. The results of this study suggest that KSHV vIRF3 plays a crucial role in KSHV-induced malignancy.

VEGF-C/VEGFR-3 signaling plays a central role in lymphatic development, regulating the budding of lymphatic progenitor cells from embryonic veins and maintaining the expression of PROX1 during later developmental stages. However, how VEGFR-3 activation translates into target gene expression is still not completely understood. We used cap analysis of gene expression (CAGE) RNA sequencing to characterize the transcriptional changes invoked by VEGF-C in LECs and to identify the transcription factors (TFs) involved. We found that MAFB, a TF involved in differentiation of various cell types, is rapidly induced and activated by VEGF-C. MAFB induced expression of PROX1 as well as other TFs and markers of differentiated LECs, indicating a role in the maintenance of the mature LEC phenotype. Correspondingly, E14.5 Mafb−/− embryos showed impaired lymphatic patterning in the skin. This suggests that MAFB is an important TF involved in lymphangiogenesis.

Several lymphatic reporter mouse lines have recently been developed to significantly improve imaging of lymphatic vessels. Nonetheless, the usage of direct visualization of lymphatic vessels has not been fully explored and documented. Here, we characterized a new Prox1-tdTomato transgenic lymphatic reporter mouse line, and demonstrated how this animal tool enables the researchers to efficiently assess developmental, surgical and pathological lymphangiogenesis by direct visualization of lymphatic vessels. Moreover, we have derived embryonic stem cells from this reporter line, and successfully differentiated them into lymphatic vessels in vivo. In conclusion, these experimental tools and techniques will help advance lymphatic research.

Schlemm's canal is an important structure of the conventional aqueous humor outflow pathway and is critically involved in regulating the intraocular pressure. In this study, we report a novel finding that prospero homeobox protein 1 (Prox-1), the master control gene for lymphatic development, is expressed in Schlemm's canal. Moreover, we provide a novel in vivo method of visualizing Schlemm's canal using a transgenic mouse model of Prox-1-green fluorescent protein (GFP). The anatomical location of Prox-1⁺ Schlemm's canal was further confirmed by in vivo gonioscopic examination and ex vivo immunohistochemical analysis. Additionally, we show that the Schlemm's canal is distinguishable from typical lymphatic vessels by lack of lymphatic vessel endothelial hyaluronan receptor (LYVE-1) expression and absence of apparent sprouting reaction when inflammatory lymphangiogenesis occurred in the cornea. Taken together, our findings offer new insights into Schlemm's canal and provide a new experimental model for live imaging of this critical structure to help further our understanding of the aqueous humor outflow. This may lead to new avenues toward the development of novel therapeutic intervention for relevant diseases, most notably glaucoma.

Lymphatic endothelial cells (LECs) are differentiated from blood vascular endothelial cells (BECs) during embryogenesis and this physiological cell fate specification is controlled by PROX1, the master regulator for lymphatic development. When Kaposi sarcoma herpes virus (KSHV) infects host cells, it activates the otherwise silenced embryonic endothelial differentiation program and reprograms their cell fates. Interestingly, previous studies demonstrated that KSHV drives BECs to acquire a partial lymphatic phenotype by upregulating PROX1 (forward reprogramming), but stimulates LECs to regain some BEC-signature genes by downregulating PROX1 (reverse reprogramming). Despite the significance of this KSHV-induced bidirectional cell fate reprogramming in KS pathogenesis, its underlying molecular mechanism remains undefined. Here, we report that IL3 receptor alpha (IL3Rα) and NOTCH play integral roles in the host cell type-specific regulation of PROX1 by KSHV. In BECs, KSHV upregulates IL3Rα and phosphorylates STAT5, which binds and activates the PROX1 promoter. In LECs, however, PROX1 was rather downregulated by KSHV-induced NOTCH signal via HEY1, which binds and represses the PROX1 promoter. Moreover, PROX1 was found to be required to maintain HEY1 expression in LECs, establishing a reciprocal regulation between PROX1 and HEY1. Upon co-activation of IL3Rα and NOTCH, PROX1 was upregulated in BECs, but downregulated in LECs. Together, our study provides the molecular mechanism underlying the cell type-specific endothelial fate reprogramming by KSHV.

Kaposi's 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 Kaposi's 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.