2001 — 2010 |
Tellides, George |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Core--Microsurgery
The Microsurgery Core will provide expertise and quality control over animal transplantation models for[unreadable] Projects by Tellides, Pober, Min, and Bender of the program. The core unit will serve as a central resource to utilize the human artery transplantation models in immunodeficient mouse chimeras that have been developed at Yale[unreadable] University. Double-mutant severe combined immunodeficient (SCID)/beige mice are grafted with human or[unreadable] synthetic arteries and are subsequently immunologically reconstituted with an adoptive transfer of human[unreadable] leukocytes and/or are treated with human cytokines, such as the species-specific Th1 factor, interferon-gamma[unreadable] (IFN-y). The interactions of the leukocytes or cytokines with the graft vascular cells results in immunemediated[unreadable] arterial injury in a surrogate human experimental model. Additionally, various mouse recipient[unreadable] strains are grafted with mouse aorta segments to take advantage of the power of murine genetic models to[unreadable] supplement the human tissue data. The Microsurgery Core will also develop and adapt the artery graft[unreadable] models according to Project requirements, e.g. developing a Rag1 mutant rat recipient to study remodeling[unreadable] in larger primary branches of human epicardial coronary arteries. All four projects will be supported by the[unreadable] Microsurgery Core. The aims of the Microsurgery Core are:1) to provide the complex small animal[unreadable] transplantation models to the Program investigators; 2) to develop new methods for improving or adapting[unreadable] the in vivo models; and 3) to provide a microsurgery training resource for investigators in vascular and[unreadable] transplantation biology. The methodology of the human artery-SCID/beige mouse transplantation model is[unreadable] relatively complex and requires shared facilities and special skills. The Microsurgery Core personnel have[unreadable] extensive experience with the required techniques and their combined expertise is essential to ensure[unreadable] consistency of the models and an economy of scale. By providing the artery graft models to all of the[unreadable] projects, the Microsurgery Core will play a key role in this program.[unreadable]
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1 |
2001 — 2005 |
Tellides, George |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Ifn Gamma in Human Ga
IFN-gamma in Human Graft Arteriosclerosis is the major limitation of cardiac transplantation and is characterized by pathological remodeling and dysfunction of coronary arteries, termed graft arteriosclerosis (GA). The pathogenesis of GA is poorly understood, but is likely immune- mediated and may result from chronic delayed type hypersensitivity (DTH) responses by recipient T cells to donor vascular antigens. Activated T cells induced DTH through secretion of cytokines, such as interferon-gamma (IFN-gamma). Paradoxically, IFN-gamma is generally thought to have an anti-proliferative effect on vascular smooth muscle cells (VSMCs) and considered to function as a pro-arteriosclerotic agent solely because of its immunomodulatory effects on vascular cells and infiltrating leukocytes. However, we have recently reported that IFN- gamma elicits arteriosclerosis in the absence of leukocytes. Our observations have led us to hypothesize that IFN-gamma acts directly on VSMCs to potential platelet-derived growth factor (PDGF)-BB induced mitogenesis through interactions involving growth factor receptors and STAT proteins. To test our hypothesis with the experiments planned in this project, we have formed productive collaborations with other investigators of the program application and together we have developed novel models of GA by establishing methods to transplant human and pig coronary arteries to immunodeficient mouse hosts and by defining conditions for the long-term organ culture of human and pig coronary arteries. We will use these approaches to elucidate the effects of IFN- gamma on vascular tissues in vivo and in vitro. The aims of our application are: (1) to test the hypothesis that IFN-gamma growth stimulatory signals to VSMCs are mediated by STAT3 and are enhanced by PDGF-BB; (2) to test the hypothesis that IFN-gamma growth inhibitory signals to VSMCs are mediated by STAT1 and are diminished by PDGF-BB; (3) to test the hypothesis that IFN-gamma induced proliferation of VSMCs is independent of IFN-gamma effects on endothelial cells; and (4) to validate our model by confirming that the expression of certain IFN gamma-dependent gene products correlates with the presence and degree of GA in human cardiac allografts the outcomes of these studies will provider considerable new information about the role of IFN-gamma in GA. Our experimental work performed in conjunction with Cores B and C will provide a mechanistic extension to the studiers of Project 1. The reagents we characterize in conjunction with Cores D and E will be applied in imaging studies by Project 3. The comparisons between experimental and clinical specimens in conjunction with Project 3, Cores D and E will validate our models. Our findings may ultimately have diagnostic, prognostic, and therapeutic clinical utility.
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2006 — 2010 |
Tellides, George |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Ifn-Gamma, Smooth Muscle Cells and Graft Arteriosclerosis
Chronic rejection, the major limitation of cardiac transplantation, is characterized by pathological remodeling and dysfunction of coronary arteries, termed graft arteriosclerosis (GA). The pathogenesis of GA is poorly understood, but is likely immune-mediated and may result from chronic delayed-type hypersensitivity responses by recipient T cells to donor vascular antigens through the secretion of cytokines, such as interferon-gamma (IFN-gamma). Paradoxically, IFN-gamma is generally thought to have an antiproliferative effect on vascular smooth muscle cells (VSMCs) and was considered to function as a proarteriosclerotic agent solely because of its immunomodulatory effects on endothelial cells and infiltrating leukocytes. However, we have found that IFN-gamma elicits arteriosclerosis in the absence of leukocytes. Our prior observations and current preliminary studies have led us to hypothesize that IFN-gamma induces VSMC proliferation that depends on a mTOR/p70S6K pathway, sensitizes VSMCs to apoptosis through upregulation of XAF1 and Noxa, and primes VSMCs for innate immune responses to fragmented nucleic acids by induction of RIG-I and MDA5. These disparate effects of IFN-gamma on VSMC survival and inflammation interact and cause intimal expansion, outward vascular remodeling, and vasodysfunction of conduit coronary arteries which ultimately determine lumen size and blood flow. We further hypothesize that these direct actions of IFN-gamma on VSMCs will be inhibited by peroxisome proliferator-activated receptor (PPAR)gamma ligands. To test our hypotheses with the experiments planned in this project, we have formed productive collaborations with other investigators of the program application and together we have developed novel models of GA in which human coronary arteries are interposed in severely immunodeficient mouse hosts that produce human IFN-gamma by adenoviral vector infection. Our methods are supplemented by mouse artery transplantation models and by cellular and molecular studies of human VSMCs. We will use these approaches to elucidate the effects of IFN-gamma on arterial tissue in vivo and in vitro. The outcomes of these studies will provide considerable new information about the role of IFN-gamma in GA, may identify novel therapeutic targets to treat and image GA, and investigate mechanisms of inhibiting GA by existing pharmacological agents.
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2013 |
Pober, Jordan S (co-PI) [⬀] Saltzman, W Mark [⬀] Tellides, George |
R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
Bioengineered Sirna/Nanoparticles to Prevent Human Transplant Rejection
DESCRIPTION (provided by applicant): We propose to apply methods and insights of bioengineering and human immunology to a surgical therapy, namely organ transplantation. Our goal is to produce safe, efficient, selective and sustained knock down of immunostimulatory proteins within human graft endothelial cells (EC) by developing ex vivo targeted nanoparticle transfection of siRNA so as to reduce allograft rejection in humanized pre-clinical models. Rejection remains an important cause of graft loss and current regimens of host immunosuppression produce significant complications. Our novel approach will reduce rejection instead by modifying the alloantigenicity of the graft. By focusing on human-based models, we address two fundamental limitations of most rodent transplant models. First, adult humans, but not experimental rodents, have circulating effector memory T cells capable of directly recognizing non-self-major histocompatibility complex (MHC) molecules and, upon activation, causing graft rejection. The high frequency of alloreactive memory cells is thought to account for the failure in humans of many therapies successful in rodents. Second, human endothelial cells (ECs), unlike rodent ECs, express and directly present non-self-class II MHC molecules to circulating effector memory T cells, initiating rejection and bypassing the need for graft dendriti cells (passenger leukocytes) to activate na¿ve host T cells seen in rodent models. Our experiments with cultured human ECs and with humanized mouse models of allograft rejection have revealed crucial roles for EC-expressed co-stimulators and EC- derived cytokines as well as EC-expressed MHC molecules in T cell activation. Furthermore, human effector memory T cells are still somewhat plastic and can be irreversibly directed along different pathways by their initial contact with graft ECs. In other words, changes in the expression of immune stimulatory or regulatory molecules by ECs in the perioperative period can have lasting effects on graft outcomes. In current clinical practice, the graft vasculature is flushed with an organ preservation solution so that ECs throughout the graft come in contact with the perfusate. We will optimize conditions for ex vivo delivery of siRNAs using biodegradable polymer nanoparticles engineered to efficiently transfect ECs lining human blood vessels and to produce a more sustained change in the EC phenotype achieved by current transfection approaches (specific aim 1); we will use this approach to knock down specific immunomodulatory molecules, examples being CIITA, LFA-3, raptor and/or IL-1a, in cultured human ECs and assess effects on the activation of allogeneic memory T cells in vitro, compared to conventional EC transfections (specific aim 2); and we will use nanoparticle- mediated transfection to knock down molecules identified as important in aim 2 in the ECs lining human artery segments ex vivo prior to implantation into mice reconstituted with a human immune system allogeneic to the artery donor, assessing the effect on acute and subacute graft rejection (specific aim 3). These pre-clinical studies will provide proof of concept for our novel approach to improve the outcome of allotransplantation.
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2017 |
Humphrey, Jay D. (co-PI) [⬀] Tellides, George |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Characterization of Tgfb-Dependent Mechanoresponses by Aortic Smooth Muscle Cells
PROJECT SUMMARY Thoracic aortic aneurysms (TAAs) affect young and old males and females and are responsible for significant morbidity and mortality. Findings over recent years suggest that an aberrant activity of or signaling through transforming growth factor-beta (TGF?) plays important roles in TAAs, yet controversy remains regarding the precise mechanisms. This lack of understanding continues to hinder the identification of improved therapeutic approaches as revealed by the recent failure of a highly anticipated clinical trial of losartan, an angiotensin-II receptor antagonist. We and others recently hypothesized that the collection of predisposing genetic mutations suggests that TAAs result from a compromised cellular mechanosensing and mechanoregulation of the extracellular matrix that endows the aortic wall with its structural integrity. Importantly, TGF? can be viewed, in part, as an important mechanotransducer ? its production and activation are mechanosensitive and its downstream gene products include the contractile proteins that are fundamental to sensing and regulating the extracellular matrix that is produced in response to its increased signaling. The goal of our work is to test novel hypotheses on interactions among the structural and instructional roles of altered TGF? signaling, smooth muscle cell mechanosensing of altered wall stresses (particularly those due to hypertension, a primary risk factor for TAAs), and the integrity of fibrillin-1, an essential glycoprotein that associates with elastin to form elastic fibers. Towards this end, we will use a combination of new genetically modified mouse models, in vivo models of induced hypertension, and clinical specimens of TAAs. Specifically, we will characterize responses of smooth muscle cells in the thoracic aorta to increased wall stresses and disrupted fibrillin-1 that depend on TGF? signaling and lead to maladaptive remodeling of the aortic wall. The results of our work will thereby provide the first mechanistic investigation of roles of TGF? signaling in cases of hypertension (a major risk factor for TAAs) and compromised extracellular matrix (fibrillin-1, the cause of the majority of syndromic TAAs) while testing, for the first time, the recently proposed hypothesis that dysfunctional smooth muscle mechanosensing and mechanoregulation of matrix underlies many different causes of TAAs. In particular, we suggest that smooth muscle cells will invoke an atrophic process if they sense stresses lower than homeostatic even in cases wherein the actual stress is normal or higher than normal, which will drive the wall toward aneurysmal development. The characterization of TGF?-dependent mechanoresponses by aortic smooth muscle cells may identify new molecular targets to treat TAAs, a lethal disease and without current pharmacotherapy.
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2017 — 2021 |
Pober, Jordan S [⬀] Saltzman, W. Mark (co-PI) [⬀] Tellides, George |
U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Ex Vivo Nanoparticle Drug Delivery Targeted to Human Renal Allograft Endothelium
A significant proportion of patients with renal failure who would benefit from kidney transplantation are highly pre-sensitized, i.e. they have high titers of pre-formed circulating antibodies (Abs) reactive with 80% or more of non-self allelic forms of class I and/or class II HLA antigens called panel reactive Abs (PRA). Upon transplantation, host PRA will bind to graft HLA antigens that are highly expressed on graft endothelial cells (ECs) where they activate host human complement resulting in deposition of membrane attack complex (MAC) on the ECs. Human MAC does not lyse human ECs, but instead alters them to express gene products that promote inflammation. The inflammatory milieu favors activation of adaptive immune effectors at the expense of protective immunoregulation. Consequently, if transplanted, patients with high titer PRA have increased episodes of acute and chronic rejection resulting in more graft failure and graft loss. Current therapeutic approaches include plasmapheresis to reduce the titer of circulating PRA, targeted elimination of Ab-producing cells and/or administration of high doses of intravenous gamma globulin to reduce inflammation, but PRA titers return and these interventions have had only limited impact on outcomes. We propose a novel strategy to complement these approaches, namely to reduce the response of graft ECs to PRA/MAC by reducing expression of HLA antigen targets and/or by inhibiting PRA/MAC signaling in ECs. The latter approach is based upon our elucidation of the relevant signaling pathways. To accomplish this, we will develop safe, polymeric nanoparticles (NPs) that are targeted towards graft ECs by means of conjugated anti-EC Abs and use these NPs to deliver siRNAs or small molecule therapeutics (?drugs?) during a period of ex vivo normothermic perfusion (EVNP), an approach that is being applied to improve energy stores in kidneys and other organs from deceased donors prior to transplantation. The NPs, which will be bound to and internalized by the graft ECs, will then serve as a depot for sustained release of the therapeutic agent for a period sufficient to allow graft accommodation and/or host immunoregulation to develop. In Specific Aim 1, we will use human EC cultures and human artery segments interposed into the aortae of immunodeficient mice to identify the optimal siRNAs or drugs that can protect ECs from PRA. Our initial target will be prevention of Akt activation, a key step in PRA/MAC signaling. In Specific Aim 2, we will identify optimal Abs for targeting renal human ECs and use these to identify conditions for efficient pan-EC delivery in human kidneys unsuitable for clinical transplantation that are subjected to EVNP by our collaborators at the University of Cambridge. (U01 support will be used only for experiments and analyses conducted at Yale; the costs of experimental EVNP will be provided by our Cambridge colleagues who are supported by a grant from the UK National Institute for Health Research and experimental EVNP will be conducted at the University of Cambridge under their Ethics Approval.) If successful, this approach can be extended to other uses and could justify a human clinical trial.
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2018 — 2019 |
Pober, Jordan S [⬀] Tellides, George |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Combined Human Myeloid and Lymphoid Engraftment in Mistrg Mice For Transplantation Research
7. Project Summary/Abstract Rodent models of organ transplantation may deviate from transplantation in the clinic both because there are species differences in basic properties of the immune system and because of extensive changes in the state of the immune system induced by encounters with microbes that do not occur in typical laboratory mice. Furthermore, the graft plays a major role in shaping the immune response and human tissue cells, especially endothelial cells (ECs), differ significantly in their immunological properties. As an important example, human CD4+ effector memory T cells are particularly effective at recognition of alloantigens displayed by human graft ECs, an interaction lacking in mice. Consequently, therapeutic strategies and reagents developed using mouse models often fail in the clinic. ?Humanized mice,? i.e. immunodeficient mice that are provided with a human immune system and then transplanted with human tissues, can address these issues and complement conventional mouse models. Humanized mice also allow testing of biologic therapeutics that often do not cross species. The principal limitation of humanized mice is that their human immune systems are incomplete, often lacking either functional T cells or functional myeloid accessory cells, cell types that must collaborate for an effective immune system. This proposal outlines an approach to improve an existing bioengineered humanized mouse model, known as MISTRG mice, that form a well differentiated myeloid compartment when inoculated as neonates with human hematopoietic stem cells (HSCs). However, MISTRG mice lack well functioning T cell responses. We propose, to remedy this by combining HSC engraftment with adoptive transfer of mature naive and/or memory T cells from the same donor as the HSCs when the mice reach adulthood. We hypothesize and will test if our approach creates a more complete human immune response without causing graft-versus-host disease (specific aim 1) and then analyze the functions of engrafted myeloid cells both on different pathways of T cell recognition of alloantigen and on T cell-mediated rejection of human skin grafts, artery grafts or synthetic tissue grafts (specific aim 2). Successful completion of these aims will provide transplantation scientists and tissue engineers with a better model to develop and test new anti-rejection strategies that can be more readily transferred to humans.
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2019 — 2020 |
Humphrey, Jay D. (co-PI) [⬀] Tellides, George |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Smooth Muscle Cell Proliferation and Degradative Phenotype in Thoracic Aorta Aneurysm and Dissection
PROJECT SUMMARY Thoracic aortic aneurysm and dissection (TAAD) are a poorly understood group of disorders responsible for significant morbidity and mortality in both sexes and all age groups, but without specific pharmacotherapy. Elucidation of disease mechanisms focus on conspicuous areas of medial smooth muscle cell (SMC) loss, whereas foci of SMC proliferation are overlooked. We found that the number of SMCs is increased in clinical specimens of TAAD, as have several other investigators. We considered that excessive SMC proliferation may exacerbate TAAD as dividing cells transition from a contractile phenotype to enter the cell cycle and daughter cells may interrupt interactions with contiguous elastic laminae or drive growth of the vessel wall. To test our hypothesis that SMC proliferation and proliferative signaling contributes to aortopathy, we developed a novel experimental model. Conditional deletion of Tsc1, a component of the tuberous sclerosis complex, in postnatal murine SMCs leads to activation of a key kinase, mechanistic target of rapamycin (mTOR), that regulates cell proliferation among other processes. Our preliminary studies reveal that induction of mTOR signaling and SMC proliferation cause progressive TAAD associated with a novel degradative phenotype of SMCs. Our goals are to understand cellular and molecular mechanisms of the disease process and to determine if relevant in other experimental models and clinical specimens of TAAD. We do not believe that the acquisition of a subset of macrophage markers and functions by degradative SMCs in the aortic media represents transdifferentiation to macrophages as recently described in atherosclerotic plaques. Rather, degradative SMCs acquire certain properties that mimic macrophage maturation, including increased protease secretion, phagocytosis, endocytosis, autophagy, and lysosome activity. Greater proteolysis, together with sequelae from loss of contractile and synthetic activity, lead to elastic fiber fragmentation and TAAD, though clearance of extracellular debris and recycling of macromolecules may retard disease progression. Our hypothesis is provocative and our preliminary data compelling. Completion of our proposed experiments will yield considerable insight into the pathogenesis of TAAD and other mTOR-dependent arteriopathies, such as atherosclerosis and aortic stiffening, and discover new therapeutic targets for this lethal disease.
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2021 |
Simons, Michael Tellides, George |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Vascular Smooth Muscle Cell Heterogeneity and Disease
Project Summary Vascular cell heterogeneity is a fascinating but poorly understood phenomenon. Numerous vascular cell types undergo fate transitions under pathological conditions. This includes endothelial cells (ECs) undergoing endothelial-to-mesenchymal transition (EndMT) and acquiring certain characteristics typical of macrophages and smooth muscle cells (SMCs). SMCs, in turn, can also acquire macrophage-like features while bone marrow-derived mononuclear cells can express certain EC and SMC markers when present at sites of chronic inflammation. These cells fate transitions have been linked to various pathologies including atherosclerosis, aneurysms, pulmonary hypertension and cavernous cerebral malformations. While the existence of these cell fate transitions is now well accepted, little is known about the origin and characteristics of SMCs undergoing these fate changes. Our preliminary data suggest that certain subpopulations of normal SMCs are particularly prone to phenotypic modulation and are predominately pathogenic. We hypothesize that a small subpopulation of normal SMCs is responsible for pathogenesis of CV diseases associated with expansion of the SMC pool and that targeting these cells might prove to be a better and more specific therapeutic approach. It is our goal in this application to define these cell populations, determine what drives their pathogenic responses and begin identifying therapeutic approaches to controlling CV illnesses driven by specifically targeting these SMC subsets.
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