Browsing by Subject "Endothelial Cells"
Now showing 1 - 6 of 6
- Results Per Page
- Sort Options
Item ApoE, ApoER2, and the Regulation of Endothelial Cell Function(2013-04-02) Ulrich, Victoria; Thorpe, Philip E.; Yanagisawa, Hiromi; Herz, Joachim; Shaul, Philip W.; Mineo, ChiekoCardiovascular disease risk is greater in individuals with the apolipoprotein E (ApoE) allele ApoE4 versus ApoE3, and this is not explained by differences in lipid profiles. A point mutant of the ApoE receptor ApoER2, ApoER2-R952Q, is also associated with greater cardiovascular disease risk, suggesting a role for ApoER2 in vascular health and disease. However, how ApoE, ApoER2, and their variants influence cardiovascular health and disease is poorly understood. We discovered in cultured endothelial cells that ApoE3 activates endothelial NO synthase (eNOS) and thereby stimulates endothelial cell migration, and that both processes require ApoER2. In contrast, ApoE4 is incapable of activating eNOS and in fact blunts ApoE3 activation of eNOS migration. In eNOS-expressing 3T3 cells, we also found that in contrast to wild-type ApoER2, ApoER2-R952Q is incapable of mediating ApoE3 activation of eNOS. Furthermore, we have determined that LDLR-/-;ApoER2-/- mice have markedly worse atherosclerosis than LDLR-/-, and in preliminary studies ApoER2-/- have impaired reendothelialization. The Overall Goal of this project is to determine how ApoE, ApoER2 and their variants influence endothelial cell function, and the implications of these processes in vascular health and disease. The first aim is to identify the molecular basis for ApoE-ApoER2 function in endothelial cells. The roles of adaptor proteins for ApoER2 and for kinase signaling in ApoE3/ApoER2 activation of eNOS were tested. The basis for impaired signaling by ApoE4 will also be explored. The second aim is to determine how ApoE and ApoER2 influence endothelial cell phenotype in vivo. Carotid artery reendothelialization after perivascular electric injury were compared in wild-type, human ApoE3-expressing, and ApoE4-expressing mice. The third aim is to determine the role of endothelial cell ApoER2 in atheroprotection. We have successfully created floxed ApoER2 mice, and atherosclerosis development will be evaluated in crosses of ApoER2flox/flox with endothelial cell-expressing Cre and LDLR-/- mice in the future. From the unique perspective of apolipoprotein signaling in endothelium, the proposed work will enhance our fundamental understanding of ApoE and ApoER2 in cardiovascular health and disease.Item Molecular Basis of HDL-Mediated Endothelial Cell Migration and Reendothelialization(2005-12-20) Seetharam, Divya; Shaul, Philip W.Vascular disease risk is inversely related to circulating levels of high density lipoprotein (HDL) cholesterol. The atheroprotective nature of HDL is attributed mainly to its role in reverse cholesterol transport (RCT). However, recent reports of human and animal studies have suggested that the atheroprotective nature of HDL is not sufficiently explained by RCT. Therefore, the mechanisms by which HDL provides vascular protection are unclear. The disruption of endothelial monolayer integrity is an important contributing factor in multiple vascular disorders, and vascular lesion severity is tempered by enhanced endothelial repair. In these studies we show that HDL stimulates endothelial cell migration in vitro in a nitric oxide-independent manner via scavenger receptor B type I (SR-BI)-mediated activation of Rac GTPase. This process does not require HDL cargo molecules, and it is dependent on the activation of Src kinases, phosphatidylinositol 3-kinase, and p44/42 mitogen-activated protein kinases. Rapid initial stimulation of lamellipodia formation by HDL via SR-BI, Src kinases and Rac is also demonstrable. Paralleling the in vitro findings, carotid artery reendothelialization following perivascular electric injury is blunted in apolipoprotein A-I null (apoA-I-/-) mice, and reconstitution of apoA-I expression rescues normal reendothelialization. Furthermore, reendothelialization is impaired in SR-BI-/- mice. Thus, HDL stimulates endothelial cell migration via SR-BI-initiated activation of Rac GTPase, and HDL and SR-BI promote reendothelialization in vivo, revealing that signaling by the HDL-SR-BI tandem has a potent beneficial impact on the cardiovascular system.Item The Organization, Structure, and Role of Endothelial Cells During Organogenesis(2019-05-28) Daniel, Edward; Buszczak, Michael; Cleaver, Ondine; Carroll, Thomas J.; Dellinger, Michael T.Cellular crosstalk is essential for proper organogenesis. Although originally assumed to be simple conduits for blood, endothelial cells (ECs) actively coordinate organ development through communicating with nearby cell types to generate properly patterned tissues. Despite extensive studies detailing the signaling pathways important for organ development, little is known about how exactly ECs influence these processes. Furthermore, attempts to study ECs in this context have been hampered, in part, by an incomplete understanding of normal endothelial development in specific organs. Here, I characterized the organization, structure, and function of ECs during organ development in the kidney and lung. I first began with an in depth anatomical analysis of the kidney vasculature over the course of fetal development. I showed that the vasculature grows coordinately with the developing epithelium and stroma from the onset of kidney development and that it forms highly stereotyped structures around these other populations. I then carried out transcriptomic analysis of renal ECs and identified many novel EC-enriched genes expressed in the fetal kidney. From this analysis, I focused on the retinoic acid (RA)-catabolizing enzyme, Cyp26b1, which is enriched in ECs in both the kidney and the lung throughout development. Surprisingly, deletion of Cyp26b1 abrogated development of the lung, but not the kidney. Cyp26b1-null lungs exhibited defects in differentiation of the distal epithelium, leading to neonatal lethality. Transcriptomic and functional analyses suggested that the phenotype observed is due to RA-dependent and RA-independent mechanisms. On the other hand, the kidney appeared to be protected from loss of Cyp26b1 by upregulation of Dhrs3 to maintain normal RA levels. Bypassing Dhrs3 by exogenously administering RA interrupted kidney epithelial development only in mice lacking at least one allele of Cyp26b1. This body of work highlights the importance of EC-mediated cellular communication during development and identifies organ-specific mechanisms that regulate this crosstalk.Item The Role of the WWTR1(TAZ)-CAMTA1 Gene Fusion in Epithelioid Hemangioendothelioma(2021-05-01T05:00:00.000Z) Driskill, Jordan Harrison; Dellinger, Michael T.; Cleaver, Ondine; McFadden, David G.; Pan, DuojiaEpithelioid hemangioendothelioma (EHE) is a devastating and mysterious vascular cancer which has no known definitive treatment. Due to a lack of valid animal or cell-based models of EHE, progress toward understanding and treating this cancer has been severely limited. However, recent studies have determined that 90% of patients exhibit a lone, characteristic in-frame gene fusion, TAZ(WWTR1)-CAMTA1. While expression of the TAZ-CAMTA1 fusion protein has been validated as a biomarker of EHE, it remains unknown whether this genetic abnormality is a passenger or a driver of EHE. In this project, I present the first genetically-engineered mouse model (GEMM) of EHE, showing that the expression of the TAZ-CAMTA1 protein in endothelial cells is sufficient to drive the formation of EHE-like tumors in the lungs of mice. Furthermore, I demonstrate that the cessation of TAZ-CAMTA1 expression leads to the regression of these vascular tumors. I also demonstrate that TAZ-CAMTA1 transforms the MS1 endothelial cell line and that subcutaneous transplantation of these cells into nude mice leads to the formation of solid, progressive EHE-like vascular tumors that have the capacity to metastasize to the lung. Utilizing these two novel models of EHE, I unravel the gene program of TAZ-CAMTA1 and demonstrate that TAZ-CAMTA1 drives a gene signature similar to TAZ, the key effector of the Hippo pathway. Expression of an activated TAZ in endothelial cells is also sufficient to drive EHE-like vascular tumors in mice, and genetic blockade of the transcriptional partners of TAZ, the TEAD family of transcription factors, prevents the formation of TAZ-CAMTA1-induced vascular tumors. Next, I show that TAZ-CAMTA1 induces an angiogenic and regenerative-like gene program in endothelial cells. I validate that TAZ-CAMTA1 exhibits gain-of-function activities by having increased resistance to proteasomal degradation and increased nuclear enrichment over TAZ. Lastly, I show that TAZ-CAMTA1 still maintains its binding to the Hippo pathway proteins which are known to negatively regulate TAZ. In summary, I generate two novel models that pinpoint TAZ-CAMTA1 as the key driver of EHE and utilize these models to suggest several new lines of investigation for the treatment of patients with EHE.Item Targeting Nanoparticles to Tumor Vasculature(2008-09-18) Marconescu, Andrei; Thorpe, Philip E.Targeting tumor vessels represents an indirect therapeutic approach in oncology by shifting the treatment away from the tumor cells themselves. Endothelial cells are generally considered genetically stable and do not use escape mechanisms against chemotherapeutic agents as frequently as tumor cells do. Also, a very large number of tumor cells can be killed by ischemia if a single vessel is occluded. Tumor vascular markers have been identified and monoclonal antibodies targeting them have been constructed in my laboratory. There are numerous approaches to make antibodies more effective in cancer treatment. One option we have investigated is to use them for liposomal targeting to tumor vessels. Nanoparticles, and liposomes in particular, are extremely versatile because they can be adapted to carry drugs, imaging agents, or energy capture agents. In my project, I have constructed liposomes targeted to three molecules identified as tumor vascular markers: VEGFR-2, phosphatidylserine (PS), and phosphatidylethanolamine (PE). To target VEGFR-2, I have used Fab' fragments derived from a series of rat monoclonal antibodies (RAFL) that bind to the extracellular domain of the receptor. For PS targeting, I used Fab' fragments derived from an anionic phospholipid binding antibody (bavituximab) and also a serum protein, beta-2-glycoprotein 1 (beta 2GP1). PE was targeted using a small antibiotic peptide, duramycin. All the liposome constructs bound to the purified target, as tested by solid phase assays. VEGFR-2 targeted liposomes bound to and were internalized by mouse endothelial cells expressing VEGFR-2. PS and PE targeted liposomes bound to endothelial cells that were subjected to stress factors that mimic the conditions encountered in the tumor environment. All the liposomes were also detected on the surface of endothelial cells inside tumors. The tumor treatment potential was assessed by loading the liposomes with doxorubicin and treating mice in an orthotopic breast cancer model. The therapeutic benefit was also assessed for its ability to prolong survival in a lung pseudometastatic model. The tumor growth in the orthotopic model was not inhibited by any of the constructs compared with control liposomes, but VEGFR-2 targeted liposomes extended the survival in the pseudometastatic model. These data suggest that VEGFR-2 targeted liposomes could potentially be used as an antimetastatic agent in combination with treatments that would target the tumor of origin. PS and PE binding liposomes were also used as probes for describing the membrane localization and exposure dynamics of PS and PE on the surface of irradiated cells. I have shown that PS and PE follow a similar exposure time course and they colocalize on the cell surface. PS and PE positive membrane patches appear to detach from the cytoskeleton and bud out from the cell surface. These findings suggest that PE and PS share common regulatory mechanisms of membrane translocation. PS and PE binding liposomes were also used as probes for describing the membrane localization and exposure dynamics of PS and PE on the surface of irradiated cells. I have shown that PS and PE follow a similar exposure time course and they colocalize on the cell surface. PS and PE positive membrane patches appear to detach from the cytoskeleton and bud out from the cell surface. These findings suggest that PE and PS share common regulatory mechanisms of membrane translocation. Long circulating liposomes provide benefit through passive targeting to the tumor environment. My findings imply that active targeting by adding a ligand should be done with care, so as not to impede the passive targeting effect. Compared to other vascular targeting agents, liposomes requireItem Vanishing Act: Lymphatic Vessels and Disappearing Bones(December 2021) Monroy, Marco Antonio; Jewell, Jenna L.; Dellinger, Michael T.; Cleaver, Ondine; Brekken, Rolf A.Generalized lymphatic anomaly (GLA) and Gorham-Stout disease (GSD) are related diseases of the lymphatic system. Patients with GLA or GSD develop ectopic lymphatic vessels in bone and gradually lose bone. Despite growing interest in the development of tissue-specific lymphatics, the cellular origin of bone lymphatic endothelial cells (bLECs) is not known, and the development of bone lymphatics has not been fully characterized. In this dissertation, I review the latest advances in research on the development of the lymphatic system and human lymphatic diseases. I also present my work on the development of bone lymphatics in mouse models of GLA and GSD. I show by lineage-tracing that bLECs arise from pre-existing Prox1-positive LECs. I demonstrate that bone lymphatics develop in a stepwise manner, where regional lymphatics grow, breach the periosteum, and then invade bone. I also show that osteoclasts are closely associated with invading lymphatics and that lymphatic invasion of bone is impaired in mice that lack osteoclasts. Additionally, I demonstrate that rapamycin suppresses the formation of bone lymphatics in mouse models of GLA and GSD. These findings shed light on the development of bone lymphatics, a process that has puzzled investigators since Gorham and Stout published their landmark paper over 60 years ago. They also show that an emerging treatment for GLA and GSD patients can inhibit lymphatic invasion of bone.