Young-Kwon Hong - US grants

[unreadable] DESCRIPTION (provided by applicant): The blood vessel-specific fluorescent transgenic mouse using the Tie2 promoter (Tie2-GFP) has enabled non-invasive real-time in vivo studies of the vascular system. In comparison, a lymphatic-specific fluorescent transgenic mouse model has not yet been established, despite its potential value. Here, we propose the generation of a transgenic mouse line that expresses the red fluorescent protein (RFP) specifically in the lymphatic system. We plan to achieve this goal by undertaking 2 separate approaches. For the first approach, we will utilize binding sites of the lymphatic specific transcription factor Prox1. In the proposal, we present our recent data of characterizing the Proxl-binding sites in the promoter of mouse fibroblast growth factor receptor (FGFR)-3, which we have identified as a Proxl target gene. We demonstrated that the Prox1-response element of 9 nucleotides (CACGCCTCT) is necessary and sufficient for the Prox1-mediated transcriptional activation of a reporter gene. Based on this finding, we will introduce a single or multiple copies of this Prox1-response element into an enhancer/promoter-less RFP reporter vector and microinject this transgene into fertilized mouse oocytes to generate a transgenic animal. In the second approach, we will take an advantage of a recently developed homologous recombination technology to knock-in the RFP gene into the Prox1 loci of 200-bk-long bacterial artificial chromosomes (BACs) that our analysis suggests contain all the regulatory elements needed for the lymphatic-specific expression of Prox1. The resulting Prox1-BAC transgenic reporter constructs will be used to generate the lymphatic-specific RFP mouse line. Furthermore, the lymphatic-specific RFP mouse line will be genetically crossed with the Tie2-GFP mouse line to establish a double transgenic mouse model whose blood and lymphatic vessels are labeled in green and red, respectively. These mouse models will be valuable tools in isolating mouse blood and lymphatic endothelial cells, to study in vivo physiological and pathological angiogenesis and lymphangiogenesis, and to readily visualize the molecular interactions of lymphatic endothelial cells with various immune cells as well as metastatic tumor cells. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Nuchal edema (NE) is a tissue swelling on the back of the neck of fetus due to an abnormal fluid accumulation during human pregnancy and has been associated with fetuses having chromosomal aneuploidy. NE can be clinically diagnosed by ultrasound and the measurement of NE, also called nuchal translucency measurement, has been widely accepted as a standard prenatal screening method to assess the risk of the fetus having chromosomal abnormalities. Studies show that more than 80% of NE fetuses carry chromosomal abnormalities such as Down syndrome. Although NE has been reported to be associated with disturbed development of the lymphatic system, the molecular basis of NE is currently unknown. In this proposal, we provide our preliminary evidence that Notch signal is dysregulated in the lymphatic system of human trisomy 21 Down syndrome fetuses and their mouse model, trisomy 16 mice embryos that exhibit NE. Previous studies show that Notch signal plays an essential role in specifying arterial cell fate. Consistently, we found that Notch can reprogram lymphatic endothelial cells (LECs) to abort the normal lymphatic differentiation program and adopt the arterial endothelial cell fate. Interestingly, a similar abnormal arterialization has been reported to occur in the venous compartment of knockout mice for the nuclear receptor COUP-TFII, a finding that indicates the key role of COUP-TFII in Notch repression in the veins. We also discovered that two Down syndrome-associated genes DSCR1 and Dyrk1a, when ectopically expressed in primary LECs, down-regulate COUP-TFII. Based on these data, we now build a novel model for the molecular basis of NE: Increased gene dosage of DSCR1 and Dyrk1a results in down-regulation of COUP- TFII and subsequent activation of Notch signal in LECs of Down syndrome fetuses, and the dysregulated Notch signal induces a pathological arterialization of the developing lymphatics, which fails embryonic tissue fluid homeostasis, causing NE. In this proposal, we propose to further dissect the molecular mechanism underlying the pathological arterialization of developing lymphatics. The outcome of our proposed studies will not only advance our current understanding of the molecular mechanism underlying the developmental programs for the arterio-venous-lymphatic endothelial cell fate specification, but also provide important insights into other vascular defects during human development.

DESCRIPTION (provided by applicant): Lymphatic circulation insufficiency, clinically manifested as lymphedema, is the most common post- operative complication among breast cancer patients and significantly compromises their quality of life. Breast cancer-associated lymphedema is caused by lymphatic obstruction due to lymph node dissection performed for tumor staging and prognosis, and post-operative irradiation therapy for cancer treatment. Damaged lymphatics fail to properly transport interstitial fluids, immune cells and tissue turnover molecules, all of which accumulate in the affected area, eventually causing a disfiguring and painful tissue swelling. Reported incidence of breast cancer-associated lymphedema ranges from 6% to 50%, depending on surgery types, follow-up treatments, obesity and number of resected lymph nodes. Importantly, as the breast cancer survival rate increases each year, more patients are expected to develop and suffer from lymphedema. Current challenges in managing breast cancer-related lymphedema include the lack of standardized diagnostic criteria, unpredictable onset of clinical symptoms, and the lack of effective treatments addressing the underlying molecular etiology other than physical compression. The goal of this study is to identify a molecular sign of developing lymphedema that can precisely predict and diagnose lymphatic insufficiency before the clinical symptoms appear. We propose to characterize the local free hyaluronan content in the affected tissues as a bio-signature that will not only help to set up standardized diagnostic criteria, but also to predict whether and when the disease occurs. In addition, to further extend our recent study demonstrating the therapeutic efficacy of retinoic acids (RAs) in lymphatic regeneration and lymphedema, we aim to optimize and improve the therapeutic protocol of RA to help treat lymphedema. By innovatively combining the hyaluronan-assisted early detection and RA-based molecular therapies, we aim to develop a prototype early intervention system against breast cancer-related lymphedema, and to establish an experimental foundation for potential future clinical use of free hyaluronan as a biomarker and RAs as therapeutic agents to treat human lymphedema patients.

DESCRIPTION (provided by applicant): The lymphatic system plays the major role in tissue fluid homeostasis by draining the interstitial fluid back to the circulation. Lymphedema, caused by lymphatic malformation or obstruction, is often associated with radiation and surgery; however effective treatments that address the underlying molecular pathology are not available to date. We have recently reported that 9-cis retinoic acid (RA) can activate cell proliferation, migration and tube formation of lymphatic endothelial cells (LECs), stimulate lymphangiogenesis in vivo, and ameliorate secondary lymphedema by promoting lymphatic regeneration in a mouse model. These pro-lymphangiogenic features of 9-cisRA, however, are quite unexpected, because RAs have been known for their anti-proliferative effects on many cell types, including blood vascular endothelial cells (BECs); where RAs have been shown to suppress BEC proliferation, and RA-deficient mouse embryos display hyper-proliferation of BECs. In this proposal, therefore, we aim to address two main questions (1) what is the molecular mechanism underlying RA-induced lymphangiogenesis, and (2) how can RAs selectively induce lymphangiogenesis, while concurrently suppressing angiogenesis. Our preliminary studies revealed that RAs may regulate Notch pathway to promote lymphatic sprouting, suggesting novel crosstalk between the two important morphogenic signals, and also that Prox1, the master regulator of lymphatic differentiation and development, can physically and functionally interact with a RA-binding nuclear receptor RXR in a RA- controlled manner. Furthermore, LECs predominantly express FABP4 as a cytoplasmic RA-carrier, and PPAR? as a dimerization partner of RXR, which is known to promote cell proliferation in response to RAs, whereas BECs selectively express CRABP-II and RAR?, a molecular pairing that induces cell growth arrest in response to RAs. Together, we propose working hypotheses addressing our two main questions that (1) RAs stimulate lymphatic sprouting by modulating Notch pathway genes through regulation of the interactions of Prox1 and RXR in LECs and (2) the predominant expression of FABP4 and PPAR? in LECs converts RA from an anti-proliferative signal to a pro-growth cue in LECs. Here, we aim to validate these working hypotheses by studying the role of RAs in promoting lymphangiogenesis through RXR? and PPAR? (Aim1), mechanism underlying the opposing effects of RAs on angiogenesis vs. lymphangiogenesis (Aim 2), and RA-controlled physical and functional interactions between Prox1 and RXR? (Aim 3). Together, our studies will not only provide important information on how Prox1 functions as the master regulator of lymphatic development by functioning as a nuclear receptor coregulator, but also define the molecular mechanism underlying RA-mediated selective promotion of lymphangiogenesis. In the long run, our study will help lay an essential experimental foundation to repurpose RAs as potential therapeutic agents for lymphatic circulation insufficiency.

PROJECT SUMMARY OBJECTIVE: We will determine the molecular mechanism by which the aqueous humor outflow (AHO) directs the formation of Schlemm?s canal (SC) through Klf4. SC is a specialized vascular structure that drains the aqueous humor from the anterior chamber into the circulation, and plays a key role in regulating the intraocular pressure (IOP). Dysfunctional SC due to aging or diseases could critically elevate the IOP and often causes ocular nerve damage, possibly leading to glaucoma. Better understanding of the reciprocal interaction between SC development and the AHO would thus have a transformative impact on the prevention of glaucoma caused by elevated IOP. RATIONALE: Structurally, SC is directly connected to the aqueous vein to drain the aqueous humor. Because of this direct vascular joining, SC has long been thought to be a specialized venous extension, whose inner wall is lined by blood vascular endothelial cells. Interestingly, however, several studies have demonstrated evidences, which distinguish SC from typical blood vessels and re-categorize SC as a new lymphatic-like vascular structure. These studies have prompted us to carefully re-examine the molecular and cellular features of SC. As the results, we and others have recently uncovered that SC is postnatally derived from the limbal vascular plexus by upregulating Prox1, the master regulator of lymphatic development. Importantly, this lymphatic reprograming of blood vessel endothelial cells (BECs)-to-SC endothelial cells (SCECs) appeared to be triggered and maintained by the optimal AHO. Accordingly, our main question to address here is how the mechanical signal from the AHO regulates the genetic program that specifies the SCEC identity. STRATEGY & GOAL: In addition to our lymphatic-specific fluorescent mouse model that was published recently, we have created a transgenic rat model whose lymphatic vessels and SC are genetically labeled with GFP. From these two novel murine models, we will purify and culture SCECs in vitro for various molecular and cellular characterizations of rodent SCECs. We have recently reported that Klf4, a shear stress responsive transcription regulator, is highly expressed in the SC precursor cells, and that Klf4 physically interacts with Prox1. Accordingly, we hypothesize that the fluid flow-induced mechanical signal may be incorporated via Klf4 into Prox1-mediated cell fate specification program, which together controls differentiation of limbal blood vessel BECs to SCECs. In Aim1, we will elucidate the mechanism of flow-mediated SCEC-fate specification through Klf4 using purified SCECs. As a preliminary study, we successfully isolated mouse SCECs and confirmed the presence of two unique ultrastructures of SCECs, namely giant vacuole and trans-cellular pores. We will study the roles of Klf4 in various molecular and cellular characteristics of isolated SCECs. In Aim2, we will employ two cohorts of tissue-specific, inducible Klf4 knockout mouse models to study the contribution of Klf4 to the initial stage of the SC organogenesis. Together, our proposed studies will provide a unique capability to address important unanswered questions on SC development, and generate valuable information to better understand the functional interaction between SC development and the IOP control with a possible therapeutic implication toward glaucoma.

PROJECT SUMMARY OBJECTIVE: We will determine the molecular mechanism by which the aqueous humor outflow (AHO) directs the formation of Schlemm?s canal (SC) through Klf4. SC is a specialized vascular structure that drains the aqueous humor from the anterior chamber into the circulation, and plays a key role in regulating the intraocular pressure (IOP). Dysfunctional SC due to aging or diseases could critically elevate the IOP and often causes ocular nerve damage, possibly leading to glaucoma. Better understanding of the reciprocal interaction between SC development and the AHO would thus have a transformative impact on the prevention of glaucoma caused by elevated IOP. RATIONALE: Structurally, SC is directly connected to the aqueous vein to drain the aqueous humor. Because of this direct vascular joining, SC has long been thought to be a specialized venous extension, whose inner wall is lined by blood vascular endothelial cells. Interestingly, however, several studies have demonstrated evidences, which distinguish SC from typical blood vessels and re-categorize SC as a new lymphatic-like vascular structure. These studies have prompted us to carefully re-examine the molecular and cellular features of SC. As the results, we and others have recently uncovered that SC is postnatally derived from the limbal vascular plexus by upregulating Prox1, the master regulator of lymphatic development. Importantly, this lymphatic reprograming of blood vessel endothelial cells (BECs)-to-SC endothelial cells (SCECs) appeared to be triggered and maintained by the optimal AHO. Accordingly, our main question to address here is how the mechanical signal from the AHO regulates the genetic program that specifies the SCEC identity. STRATEGY & GOAL: In addition to our lymphatic-specific fluorescent mouse model that was published recently, we have created a transgenic rat model whose lymphatic vessels and SC are genetically labeled with GFP. From these two novel murine models, we will purify and culture SCECs in vitro for various molecular and cellular characterizations of rodent SCECs. We have recently reported that Klf4, a shear stress responsive transcription regulator, is highly expressed in the SC precursor cells, and that Klf4 physically interacts with Prox1. Accordingly, we hypothesize that the fluid flow-induced mechanical signal may be incorporated via Klf4 into Prox1-mediated cell fate specification program, which together controls differentiation of limbal blood vessel BECs to SCECs. In Aim1, we will elucidate the mechanism of flow-mediated SCEC-fate specification through Klf4 using purified SCECs. As a preliminary study, we successfully isolated mouse SCECs and confirmed the presence of two unique ultrastructures of SCECs, namely giant vacuole and trans-cellular pores. We will study the roles of Klf4 in various molecular and cellular characteristics of isolated SCECs. In Aim2, we will employ two cohorts of tissue-specific, inducible Klf4 knockout mouse models to study the contribution of Klf4 to the initial stage of the SC organogenesis. Together, our proposed studies will provide a unique capability to address important unanswered questions on SC development, and generate valuable information to better understand the functional interaction between SC development and the IOP control with a possible therapeutic implication toward glaucoma.

PROJECT SUMMARY Lymphatic valves are essential for the proper lymph flow occurring during tissue fluid drainage and transport, immune cell trafficking and nutrient absorption in the intestine. Despite their critical roles in vascular function and health, relatively little is known about the molecular mechanism that controls lymphatic valve development and maintenance. In this proposal, we aim to elucidate the mechanotransduction that incorporates the fluid flow-driven external signals into the internal genetic programs governing the valve formation and function in the lymphatic system. We have recently identified a unique fluid flow condition that could induce the signatures of the early stages of lymphatic valve development, including Prox1 upregulation, an initial hallmark of valvular endothelial cell specification that was not attainable by other reported flow conditions. Imposing mechanical cell stretching, this experimental setup induces p65 nuclear translocation, Prox1 upregulation and dephosphorylation, and their functional collaboration through both protein-protein interaction and binding site sharing. These observations have led us to hypothesize that Prox1 and p65 cooperatively control a yet uncharacterized valve-forming mechanotransduction pathway in lymphatic compartment, and possibly conserved in venous vessels as well. We propose that our experimental flow condition will allow us to address this hypothesis by offering a unique opportunity to dissect the mechanotransduction. In addition, we will confirm the regulation and function of the essential physical, molecular and genetic constituents of the mechanotransduction using various animal models. The outcome will not only advance our current understanding of lymphatic valve formation and function, but also provide fundamental insights into the mechanisms underlying the flow-regulated vascular development and function.

PROJECT SUMMARY The homeodomain transcription factor PROX1 is necessary and sufficient for induction and maintenance of lymphatic endothelial cell identity. It not only plays an essential role in the initial lymphatic reprogramming, expansion, maturation and maintenance, but also directs formation of luminal and lymphovenous valves. Although Prox1 has been intensively studied for its function, structure, and regulation, it remains unclear how such numerous developmental and environmental signals are efficiently incorporated to and regulate Prox1. The objective of this study is to gain a detailed mechanistic understanding of how lymphangiogenic signals triggered by growth factors and inflammatory cytokines are transduced to Prox1 protein in the form of phosphorylation, modulate its properties, and eventually orchestrate lymphatic development and function. We hypothesize that the RAF-ERK-RSK signal cascade mediates various lymphangiogenic signals and phosphorylates PROX1 at S79, and that this modification significantly alters the biological properties of Prox1 during lymphatic development and function. To address these hypotheses, we propose to study of the impact of phospho-S79 to the behaviors of PROX1, and to elucidate the regulation of lymphangiogenesis by the ERK-RSK2-PROX1 (S79) axis under the physiological and pathological conditions. Finally, we will generate mutant mouse models that allow a tissue- specific, conditional replacement of the endogenous wild type Prox1 with its phospho-mutants. Using these animal models, we will study of the impact of PROX1 S79 phospho-mutation to lymphatic development and function in health and disease. In summary, the proposed study will define how Prox1 S79 phosphorylation, as a biomarker of activated lymphatic vessels, regulates physiological and pathological lymphangiogenesis. The outcome of this study will not only deliver a significant impact on our current understanding of the functional mode of PROX1 as the master regulator of the lymphatic system, but also offer broader insights into vascular development and function.

PROJECT SUMMARY Kaposi's sarcoma (KS) is one of the most common cancers in HIV-positive patients, and frequently occurs in the oral mucosa, skin, and lymph nodes due to KS Herpes Virus (KSHV) infection. Proliferating KS tumor cells are believed to originate from blood vascular endothelial cells (BECs) and lymphatic endothelial cells (LECs), as KS cells express the signature genes of both cell types. LECs and BECs are closely related because LECs are derived from BECs during development, thus their gene expression profiles remain similar even after development. Despite their similarities, these two types of endothelial cells exhibit significant differences in their KSHV pathologies. One of the most recognized differences is that LECs are much more permissive to KSHV infection than BECs. Although this differential infectivity was first described more than a decade ago, neither rigorous follow-up characterization, nor elucidation of the underlying molecular basis, of this important lineage-specific phenotype are understood to date. In the current study, we propose to study the molecular underpinnings of the increased KSHV infectivity in LECs. We hypothesize that CEACAM1 and CEACAM6 proteins function as a novel KSHV receptor predominantly expressed in LECs, enabling the increased permissiveness of LECs. Accordingly, we will identify and characterize these CEACAMs (Aim1) and dissect the interactions between KSHV and these CEACAM proteins (Aim2). The outcome of this study will not only define the molecular basis of the enhanced permissiveness of LECs to KSHV infection, but also provide new knowledge on lymphatic-specific KSHV pathologies. In sum, this project will advance our current understanding of KSHV-host cell interactions, help broaden therapeutic options against KS, and offer important insight into the histogenetic origin of KS.

PROJECT SUMMARY This project is proposed to respond to Provocative Question 6 in RFA-CA-19-032. Objective: Paracrine transformation is a theoretical concept that was proposed years ago to explain the unconventional ?non-autonomous? oncogenesis observed during development of Kaposi?s sarcoma (KS), one of the most common AIDS-associated malignancies. This proposal is designed to prove its existence, to dissect its mechanism, identify the players therein, and to define its roles in KS tumorigenesis using our novel animal models and an engineered microphysiological platform. Rationale: Kaposi?s sarcoma herpes virus (KSHV) causes an endothelial cell tumor, KS, in the skin and internal organs. A paradox in KS oncogenesis is that while most KS tumor cells are latently infected with minimal viral gene expression, only lytic-stage cells express vGPCR, the only known viral oncogene that is necessary and sufficient for KS development. Provocative Question: How vGPCR, a lytic viral gene expressed in cells destined to die, can cause cancer? Challenges: This question remained unanswered due to the lack of proper animal models, engineered in vitro or ex vivo systems to study pathogenesis, persistence, and tumor development that recapitulate this HIV/AIDS- associated malignancies. Innovation & Strategy: We have developed a series of novel animal models and Vascularized Skin Chip platform. Using these technical advancements, we will prove the existence of paracrine transformation, identify its cellular (immune cells, HIV) and molecular (vGPCR-loaded exosome) players, and characterize its mechanism as the main oncogenic driver for KS tumorigenesis. Impact: Our study will address the decades-long conundrum on KS tumor development by defining the existence and mechanism of paracrine transformation. This provocative concept of paracrine transformation will not only force us to move our focus beyond the lytic-infected cells as the oncogenic drivers, but also expand the way we understand the initiation, progression, and metastasis of cancer. In addition, this study will open a new door to novel anti-KS therapeutics, and provide a solid justification to investigate the presence of equivalent non-autonomous transformation in other non-viral oncogenesis, such as breast and colon cancers.

PROJECT SUMMARY To address the widely recognized decline in the physician-scientist workforce, new approaches are needed to recruit talented physicians to pursue research as an integral part of their medical training and future careers. The Stimulating Access to Research in Residency (StARR) initiative provides a new point of access to research by accelerating the entry of residents into meaningful research pursuits during residency. The USC-StARR Program will draw the most promising Resident Investigators from the General Surgery, Integrated Vascular Surgery, and Integrated Cardiothoracic Surgery Residency Programs. We have designed an intensive, 24-month, contiguous StARR program. The goal of the USC-StARR program is to recruit, train, and mentor a group of exceptional Resident Investigators in acquiring rigorous research skills, conducting high-impact, clinically relevant research projects, and launching promising careers as surgeon-scientists in cardiovascular and pulmonary science. Our program will include two research tracks: 1) Basic and Translational Research and 2) Health Outcomes, Community Engagement, and Dissemination and Implementation Research. Our goals will be accomplished through the following specific aims: 1) recruit and train three Resident Investigators annually with the potential and commitment to become successful surgeon-scientists in cardiovascular or pulmonary science; 2) guide these Resident Investigators in obtaining more advanced methodological, analytic, and collaborative research skills appropriate for their level of training; 3) create and support effective, influential, and long-lasting mentor relationships during and after residency; and 4) guide Resident Investigators in successfully competing for other forms of clinical and translational research support that will pave the way for them to pursue long-term careers as surgeon-scientists. A Multi-Program Director structure will provide Administrative and Scientific leadership for the program. The Resident Investigators will have access to a cadre of carefully selected Research Preceptors with sustained NIH funding coupled with successful track records of mentoring early career scholars. A group of four highly dedicated and accomplished senior scientists will serve on the Advisory Committee of the program. Each Resident Investigator will work with their Research Preceptor, Advisory Committee, and the Scientific Director to achieve their individual and program goals, to provide independent evaluation of their progress, and to develop, advise on, and track their Independent Career Development Plan. The impact of this innovative proposal will be to increase the ability and capacity of the USC General Surgery, Integrated Vascular Surgery, and Integrated Cardiothoracic Surgery residency training programs to foster the development of surgeon- scientists in the field of cardiovascular and pulmonary science. Through this program, we will increase the ability of these surgeon-scientists to transition to other forms of research and career development support.