My lab currently has three project areas. These are best summarized by the Specific Aims sections of the grants that fund them:
Research Area 1: Vascular cell lineages and phenotype in adult blood vessel formation.
The formation and remodeling of new vessels in injured tissue is essential for tissue repair. In this application we propose to test the general hypothesis, suggested by our previous quantitative aggregation chimera analysis, that new vessel formation and remodeling in adult granulation tissue involves pathways of vascular cell origin and fate that differ from pathways that predominate during embryogenesis, and that different lineages provide cells that differ in kinetics, phenotype, and function.
SPECIFIC AIM 1. Test the hypothesis that significant subpopulations of the endothelial cells (ECs), smooth muscle cells (SMCs,) and (myo)fibroblasts in granulation tissue derive from sources other than proliferation/migration of pre-existing cells of the same phenotype.
We propose to test this hypothesis using transgenic mice containing different combinations of cell type-specific "marking" and phenotype-specific "conditional reporting" constructs. This system will allow us to follow the fates of different cell types that participate in response to injury and to determine to what extent they change their phenotype during this process. We will use this conditional reporter system to determine the relative contribution of cells from three possible origins: proliferation of pre-existing cells of the same cell type, immigration and differentiation of hematopoietic progenitors, transdifferentiation of other non-hematopoietic cells.
SPECIFIC AIM 2. Test the hypothesis that SMC, EC, and (myo)fibroblast subpopulations in granulation tissue that derive from different sources are not functionally equivalent, and that they differ in phenotype (transcript and protein expression) and cell kinetics (proliferation during the formation phase and apoptosis during the resolution phase).
Much attention has been paid in recent literature to alternative origins of differentiated cells. Little attention has been paid to possible differences in phenotype and subsequent behavior of cells from different origins. The different sources of vascular cells identified in Aim 1 could "merely" combine additively to supply the requisite numbers of final cells. In Aim 2 we test the alternative hypothesis that cells from different origins are functionally different and play different roles in tissue response to injury. To do this, we will: 1. Compare the kinetic behavior (proliferation, migration, apoptosis) of cells based on their origin. 2. Sort cells by lineage/fate using the conditional EGFP reporter system and use transcript array (and individual protein expression phenotype) to compare the pattern of gene expression as a function cell origin/lineage.
SPECIFIC AIM 3. Determine the cell lineage pathways through which PDGFRb regulates the participation of ECs (and other cells) in granulation tissue formation and muscle response to injury.
We will determine this using the cell type-specific regulatory system (Aim 1) to regulate a conditional knockout of PDGFRb.
Research Area 2: Regulation and consequences of vascular smooth muscle apoptosis
Unlike necrosis, apoptosis is classically considered to be "silent", ie self-contained and non-inflammatory. We have developed a system for regulating apoptosis of vascular smooth muscle cells (SMCs) in vivo, and have used this system to determine that SMC apoptosis initiated by FADD overexpression includes a specific program of expression of pro-inflammatory genes that results in recruitment of macrophages. Binding of Fas ligand (FasL) to cultured human SMCs initiates a comparable program. In this application, we propose to further define this program of gene expression, determine the mechanisms through which it is regulated, and further investigate the consequences of SMC apoptosis in vivo. To do this, we propose the following specific aims:
SPECIFIC AIM 1: Determine the long-term consequences of SMC apoptosis in the neointima.
In our work to date, we have used a system for initiating SMC apoptosis in the rat carotid artery to determine that SMC apoptosis is accompanied by upregulation of a program of pro-inflammatory gene expression, and that this results in the recruitment of macrophages into the neointima by 14 days. In Aim 1, we will evaluate the long term consequences of SMC apoptosis in the vessel wall, including the clinically important processes of vascular remodeling, thrombosis, and plaque rupture.
SPECIFIC AIM 2. Determine the complete program of Fas-activated gene expression.
Our ultimate goal is to understand the full constitution and regulation of the apoptotic SMC expression phenotype. Expression array analysis can be an efficient method for obtaining information about changes in expression of a large number of genes. We will use expression array analysis to test the hypotheses that: 1) The Fas-activated program in SMCs includes upregulation of different sets of genes at early and late times after FasL stimulation. 2) Subsets of these genes are regulated through specific signal transduction pathways activated by Fas (identified in Aim 3). 3) The Fas-activated apoptotic program in SMCs includes genes involved in matrix remodeling/degradation 4) The program of inflammatory gene upregulation during apoptosis is characteristic of connective tissue/structural cells, ie is a property of their differentiated phenotype.
SPECIFIC AIM 3. Determine the signal transduction pathway(s) through which Fas activation leads to transcript upregulation in vascular SMCs.
We will test the hypothesis that full upregulation of gene expression by Fas activation involves an early activation of a caspase-independent pathway that requires PIP3 signaling and activation of NF-kB activity, (and probably activation of other transcription factors) and a later, caspase-dependent, pathway that amplifies the response through secretion of IL-1a.
SPECIFIC AIM 4. Determine how SMCs resist Fas-induced apoptosis, and whether Fas activates pathways that lead to cell proliferation if/when the SMCs survive longer.
The general focus of this application is to determine the consequences, and mechanism of regulation, of the program of Fas-induced gene upregulation, rather than to investigate mechanisms of Fas-induced apoptosis per se. However, it is also important to understand the mechanisms that permit the SMCs to survive long enough to carry out this program. In this aim, we propose experiments to evaluate the basic components of this protection in SMCs, and whether Fas activates pathways that would lead to cell proliferation if/when the SMCs survive longer.
Research Area 3: Biochemistry and function of a novel phosphatidylinositol phosphatase
PTPRQ is a protein tyrosine phosphatase (PTPase)-like protein that we initially identified and cloned based on its upregulation in a rat model of renal injury. Overexpression of the catalytic domain of PTPRQ in cultured cells inhibits proliferation and induces apoptosis. We have found that PTPRQ is a very unusual member of the PTPase superfamily in that its biological activity is due to its activity as a phosphatidylinositol phosphatase (PIPase) rather as a PTPase. PTPRQ has broader PIPase activity than does the tumor suppressor PTEN, the first PTPase shown to have PI 3-phosphatase activity, but it has a more restricted pattern of expression. The receptor-like form of PTPRQ protein appears to be largely localized to specialized regions of non-proliferating cell types (including podocytes, inner ear hair cells and Sertoli cells) that are involved in cell-cell or cell-matrix interactions. Targeted disruption of PTPRQ in mice results in deafness and altered response to renal injury. We propose to test the general hypothesis that subcellular localization of PTPRQ in specialized membrane regions plays a role in regulation of local membrane PIP composition, and that this plays a role in regulating specialized cell architecture and function by altering the local binding and/or activity of PIP-binding proteins.
SPECIFIC AIM 1. Determine the structure and in vitro PIPase activity of PTPRQ and test the hypothesis that the PTPRQ catalytic domain defines a group of PTPase-like domains with PIPase activity.
We have already established that the soluble recombinant catalytic domain of PTPRQ has phosphatidylinositol phosphatase (PIPase) activity in vitro. In this aim, we address remaining questions about the basic structure and function of PTPRQ in simple in vitro systems: What is the 3D structure of PTPRQ that permits its PIPase activity? Is this structure generalizable to other putatively-inactive PTPase-like domains? How does the PTPRQ catalytic domain behave when brought to the membrane as part of a receptor-like protein? We will also test the general hypothesis that the putatively inactive D2 domains of a defined class of dual-domain receptor-like PTPases have PIPase activity. This would give these receptor-like PTPases dual activity: as PTPases, via their D1 domains, and as PIPases via their D2 domains. If confirmed, this hypothesis would be quite significant.
SPECIFIC AIM 2. Test the hypothesis that cleavage of specific membrane PIPs by transmembrane forms of PTPRQ regulates the subcellular localization/activation of PIP-binding proteins in specialized cells (including podocytes, Sertoli cells) and thereby regulates the spatial organization and function of those cells.
In this aim we describe experiments designed to determine the enzymatic activity and specificity of transmembrane-tethered and receptor-like forms of PTPRQ in cultured cells. We will test the hypotheses that: 1) The activity/specificity of PTPRQ will be altered by subcellular localization, and by interaction with other cellular components, including cytoplasmic and membrane proteins. 2) The PIPase activity of membrane-tethered and receptor-like forms of PTPRQ will affect cell content of PI(3,4,5)P3. This will contribute to the regulation of "general" cell pathways/responses, including proliferation and apoptosis. 3) Subcellular localization of PTPRQ, as observed in podocytes, Sertoli cells, and hair cells, will regulate the local concentrations of membrane PIPs, and this will function to localize and activate membrane-associated proteins that form or regulate specialized cell architecture and function.
SPECIFIC AIM 3. Test the hypothesis that the extracellular domain of PTPRQ binds ligands or counter-receptors, and that this regulates its activity and/or localization.
Based on its deduced structure, we hypothesize that the extracellular domain of PTPRQ interacts with a ligand or counter-receptor, and that this interaction functions to localize PTPRQ within the cell and/or to change the PIPase activity of the cytoplasmic domain. In this aim, we describe the strategy we will use to identify, clone, and use this ligand/counter-receptor.
SPECIFIC AIM 4. Evaluate the biological function of PTPRQ by determining which cells express it and what are the consequences of lack of expression in KO mice.
In this aim, we propose to further evaluate PTPRQ expression and function in the context of the whole tissue/organism. We will test the hypothesis that PTPRQ is expressed at sites of specialized cell-matrix (or cell-cell) interactions by completing our determination of where, at the cellular and subcellular level, PTPRQ is expressed in mouse and human tissues. We will evaluate the function of PTPRQ in mice by determining the consequences of loss of expression in PTPRQ-/- KO strains.