Joseph A. Madri, Ph.D, M.D - US grants

Atherosclerotic vascular disease is a major cause of morbidity and mortality in the U.S. Endothelial cell injury/dysfunction is responsible for the initiation of at least some of the disease and the endothelial cell (EC) response to injury is a major factor in determining and modulating the resultant migratory, proliferative, synthetic and contractile responses of the medial smooth muscle cells and the angiogenic response of the adventitial microvasculature. The EC response to injury, modulated by the composition and organization of the underlying extracellular matrix and endothelial cell-extracellular matrix interactions, although incompletely understood, is thought to be a dynamic, complex one involving several classes of matrix binding proteins. The long-term goal of this proposal is to elucidate the roles of integrin and non-integrin cell surface matrix binding proteins in modulating large vessel endothelial cell migration and proliferation and microvascular endothelial cell angiogenesis following injury and in response to soluble factors. Specifically, tissue culture and animal models of large vessel endothelial cell denudation injury-repair and microvascular endothelial cell angiogenesis will be used to characterize and determine the mechanism(s) of action of integrin and non- integrin matrix binding proteins during tissue culture, immunolabeling at light, confocal and electron microscopic levels, Northern, Southern and in situ hybridization and differential library screening, biosynthetic labeling, immunoprecipitation and immunoblotting. Experiments will center around the use of synthetic peptides of various binding domains of matrix molecules, antibodies raised against matrix molecules and matrix binding proteins and cRNA and cDNA probes specific for matrix molecules and matrix binding proteins in vitro large vessel endothelial cell migrations and during in vitro angiogenesis studies with microvascular endothelial cells. A better understanding of cell-matrix interactions during these processes may lead to the design, production and implementation of improved synthetic grafting materials, agents that promote optimal endothelial cell migration following therapeutic intervention and agents that can be used to modulate the angiogenic response following injury and during the metastatic spread of cancer.

Atherosclerotic vascular disease is a major cause of morbidity and mortality in the U.S. Endothelial cell injury/dysfunction is responsible for the initiation of at least some of the disease and the endothelial cell (EC) response to injury is a major factor in determining and modulating the resultant migratory, proliferative, synthetic and contractile responses of the medial smooth muscle cells and the angiogenic response of the adventitial microvasculature. The EC response to injury, modulated by the composition and organization of the underlying extracellular matrix and endothelial cell-extracellular matrix interactions, although incompletely understood, is thought to be a dynamic, complex one involving several classes of matrix binding proteins. The long-term goal of this proposal is to elucidate the roles of integrin and non-integrin cell surface matrix binding proteins in modulating large vessel endothelial cell migration and proliferation and microvascular endothelial cell angiogenesis following injury and in response to soluble factors. Specifically, tissue culture and animal models of large vessel endothelial cell denudation injury-repair and microvascular endothelial cell angiogenesis will be used to characterize and determine the mechanism(s) of action of integrin and non- integrin matrix binding proteins during tissue culture, immunolabeling at light, confocal and electron microscopic levels, Northern, Southern and in situ hybridization and differential library screening, biosynthetic labeling, immunoprecipitation and immunoblotting. Experiments will center around the use of synthetic peptides of various binding domains of matrix molecules, antibodies raised against matrix molecules and matrix binding proteins and cRNA and cDNA probes specific for matrix molecules and matrix binding proteins in vitro large vessel endothelial cell migrations and during in vitro angiogenesis studies with microvascular endothelial cells. A better understanding of cell-matrix interactions during these processes may lead to the design, production and implementation of improved synthetic grafting materials, agents that promote optimal endothelial cell migration following therapeutic intervention and agents that can be used to modulate the angiogenic response following injury and during the metastatic spread of cancer.

The glomerular and tubular capillary units are specialized filtration apparati having complex matrix organization and cell-cell interactions. Dysfunction of these units is noted in a variety of renal diseases. While a good deal is known about glomerular podocyte and tubular epithelial cell development, junction complex formation, slit diaphragm formation, function and response to injury, much less is known about the development, maintenance and the response to injury of tubular and glomerular endothelial cells (EC). In general, EC dynamics during development and in response to injury are thought to be modulated, in part, by the composition and organization of the surrounding extracellular matrix (ECM) and soluble factors in the local environment. furthermore, this dynamic interaction of EC with themselves and with ECM is thought to involve several classes of cell adhesion molecules (CAMs) such as PECAM-1, substratum adhesion molecules (SAMs) such as the integrins and cell junctional associated molecules (JAMs) such as ZO1. In this proposal we will examine the hierarchical (spatiotemporal) interrelationships of selected moieties of these three protein families in the processes of tube formation by microvascular endothelial cells in vitro and in the fetal and newborn rat kidney. Specifically, we will examine the roles of PECAM-1 in initiating and modulating the processes of in vitro and in vivo angiogenesis. The interactions of the cytoplasmic domain of PECAM-1 with elements of the filamentous and cortical membrane cytoskeleton will also be investigated. We will utilize affinity chromatography with recombinant proteins; stable transfection and over-expression of the cytoplasmic domain and truncation mutants of this PECAM-1 domain in endothelial cells; functional PECAM-1 antibodies and soluble PECAM-1 antigen in inhibition studies; and utilize immunochemical and immunohistochemical methods to assess the localizations and organization of PECAM-1 on the endothelial cell surface.

DESCRIPTION (Adapted from Investigator's Abstract): Angiogenesis during development, during healing and during tumor vascularization are processes which have major impact upon both an organism's ability to develop and survive. The control/modulation of angiogenesis is thought to involve integrated dynamic interactions among endothelial cells, extracellular matrix, soluble factors and other adjacent cell types. Advances in our understanding of these interactions in angiogenesis in the last several years have occurred in the areas of cell-cell, cell- matrix and cell-growth factor interactions. Cell-cell interactions between and among endothelial cells and endothelial cell-matrix interactions have been implicated in the various stages of the angiogenic process, including tube formation, maintenance and involution. Extracellular matrix-driven, integrin-mediated modulation of endothelial behavior has been shown to be critical in the angiogenic process. In particular, integrin modulation of Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1) phosphorylation has been shown to be an important regulator of angiogenesis both in vivo and in vitro. In particular, extracellular matrix-driven, integrin-mediated PECAM-1 tyrosine phosphorylation/dephosphorylation has been implicated in modulating endothelial cell migration, vasculogenesis and angiogenesis. Thus, the application proposes to elucidate the tyrosine and serine phosphorylation states of PECAM-1 and their effects on the conformation of the PECAM-1 ITAM domain, binding sites, and affinities for selected signalling and adapter molecules (c-src, SHPTP-2 and beta-catenin) and putative PECAM-1 - PECAM-1 interactions. Each aim is likely to provide novel information concerning PECAM-1. A particularly important aspect of this work is the use of 3D matrices to study endothelial biology. The investigator has been a leading figure in the use of the matrices. The environment is excellent for carrying out this work.

DESCRIPTION (Applicant's abstract): Our hypothesis is that adhesion molecule-dependent modulation of T cells and endothelia modulates not only adhesive functions, but also initiates specific protease induction, surface assembly, and activation which facilitates transmigration, as well as changes in adhesive properties of the T cells which affects residency of the cells at the site of inflammation. T cell transmigration through the endothelial cell layer and migration into the underlying and surrounding extracellular matrix is initiated by T cell adhesion to the endothelium, mediated by specific ligands resident on the surfaces of both the T cell [VLA-4 (a4B1)] and the endothelial cell (VCAM-1). We have demonstrated that engagement of this receptor/ligand pair evokes changes in MMP-2 expression and activation, consistent with the manifestation of an invasive phenotype in the adherent T cell population and an "activated" phenotype in the endothelial cells. Resultant proteolysis of basement membrane and interstitial matrix components is thought to facilitate T cell extravasation out of the affected vessel and toward the site of inflammation and angiogenesis. In this proposal we will: 1) determine, compare and contrast the MT1-MMP/TIMP-2/MMP-2 ternary complex characteristics in T lymphocytes and endothelial cells following their stimulation. 2) continue our characterizations of the MT1-MMP and MMP-2 promoters and their respective pertinent transcription factors. 3) identify, characterize, compare and contrast the signal transduction pathways involved in MT1-MMP and MMP-2 induction, complex formation, activation and clustering in T lymphocytes and endothelial cells. These aims will be accomplished with a combination of methodologies including an in vitro culture model utilizing antigen-specific murine T cell clones and lines; an in vivo adoptive transfer murine model of experimental allergic encephalomyelitis (EAE) and a variety of cellular and molecular biological techniques including cell culture, zymography, reverse zymography, immunoprecipitation, Western blotting, Northern blotting, transfection and stable expression of selected gene products, histology, immunohistochemistry, MALDI-TOF, DNA array analyses and the use of selected transgenic and knockout mice. These experiments will lead to a better understanding of T cell migration through and interaction with local extracellular matrix and the development of new and novel therapies directed at modulating selected proteinase/proteinase inhibitor cascade systems in the inflammatory processes of arthritis, vasculitis, and tissue rejection organ.

DESCRIPTION (provided by applicant): Our hypothesis is that there is a complex, dynamic interaction between neurotrophic receptor and "vascular" receptor signaling in neurons and endothelia in the CNS during postnatal development and it is disrupted during chronic sublethal hypoxia. During this grant period, using a variety of methodologies, we propose to: 1. Characterize the expression of neurotrophic (NT) receptors (BDNF receptors) and VEGF & angiopoietin receptors (VEGFR1 & 2, Tie2) on neurons and endothelia in the CNS (cortex) in vivo and in vitro in normoxic and hypoxic conditions. 2. Characterize the interactions and the signaling pathways involved following NT (trkB & p75NGF) and VEGF (VEGFR1 & R2) receptor engagement in neurons in vitro in normoxic and hypoxic conditions. Specifically, we will determine: the effects of VEGFR signaling upon NT receptor expression and signaling in cerebral neurons; the effects of NT signaling upon VEGF receptor expression and signaling in cerebral neurons; and the effects of sequential NT and VEGF (and VEGF and NT) receptor engagement upon neuronal proliferation, survival and differentiation. 3. Characterize the interactions and the signaling pathways involved following NT (trkB & p75NGF) and VEGF (VEGFR1 & R2) receptor engagement in cerebral endothelial cells in vitro in normoxic and hypoxic conditions. Specifically, we will determine; the effects of VEGFR signaling upon NT receptor (trkB & p75NGF) and ligand (BDNF) expression in cerebral microvascular endothelial cells; the effects of NT signaling (via BDNF) upon VEGF receptor expression and signaling in cerebral microvascular endothelial cells; and the effects of sequential NT and VEGF (and VEGF and NT) receptor engagement upon endothelial proliferation, survival and differentiation. 4. Determine NT and VE--GF receptor and ligand expression patterns and interactions in cocultures of neurons & cerebral microvascular endothelial (RBE4) cells; RBE4 cells & astrocytes; and neurons, RBE4 cells and astrocytes in vitro in normoxic and hypoxic conditions. 5. Investigate and characterize selected novel genes identified in our core facility by cDNA array analyses that are modulated during chronic hypoxia and pertinent to Aims 1 - 4. The experiments proposed in this grant application have as their goal the elucidation of the complex, dynamic interactions between neurotrophic receptor and "vascular" receptor signaling in neurons and endothelia in the CNS during postnatal development and their disruptions during chronic sublethal hypoxia. Knowledge accrued during the next grant period will facilitate the development of new and novel rational therapeutic approaches and agents which will be more specific and selective in their modulation of these cell types during the critical period of postnatal brain development in the premature newborn.

DESCRIPTION (provided by applicant): Our hypothesis is that there is a complex, dynamic interaction between neurotrophic receptor and "vascular" receptor signaling in neurons and endothelia in the CNS during postnatal development and it is disrupted during chronic sublethal hypoxia. During this grant period, using a variety of methodologies, we propose to: 1. Characterize the expression of neurotrophic (NT) receptors (BDNF receptors) and VEGF & angiopoietin receptors (VEGFR1 & 2, Tie2) on neurons and endothelia in the CNS (cortex) in vivo and in vitro in normoxic and hypoxic conditions. 2. Characterize the interactions and the signaling pathways involved following NT (trkB & p75NGF) and VEGF (VEGFR1 & R2) receptor engagement in neurons in vitro in normoxic and hypoxic conditions. Specifically, we will determine: the effects of VEGFR signaling upon NT receptor expression and signaling in cerebral neurons; the effects of NT signaling upon VEGF receptor expression and signaling in cerebral neurons; and the effects of sequential NT and VEGF (and VEGF and NT) receptor engagement upon neuronal proliferation, survival and differentiation. 3. Characterize the interactions and the signaling pathways involved following NT (trkB & p75NGF) and VEGF (VEGFR1 & R2) receptor engagement in cerebral endothelial cells in vitro in normoxic and hypoxic conditions. Specifically, we will determine; the effects of VEGFR signaling upon NT receptor (trkB & p75NGF) and ligand (BDNF) expression in cerebral microvascular endothelial cells; the effects of NT signaling (via BDNF) upon VEGF receptor expression and signaling in cerebral microvascular endothelial cells; and the effects of sequential NT and VEGF (and VEGF and NT) receptor engagement upon endothelial proliferation, survival and differentiation. 4. Determine NT and VE--GF receptor and ligand expression patterns and interactions in cocultures of neurons & cerebral microvascular endothelial (RBE4) cells; RBE4 cells & astrocytes; and neurons, RBE4 cells and astrocytes in vitro in normoxic and hypoxic conditions. 5. Investigate and characterize selected novel genes identified in our core facility by cDNA array analyses that are modulated during chronic hypoxia and pertinent to Aims 1 - 4. The experiments proposed in this grant application have as their goal the elucidation of the complex, dynamic interactions between neurotrophic receptor and "vascular" receptor signaling in neurons and endothelia in the CNS during postnatal development and their disruptions during chronic sublethal hypoxia. Knowledge accrued during the next grant period will facilitate the development of new and novel rational therapeutic approaches and agents which will be more specific and selective in their modulation of these cell types during the critical period of postnatal brain development in the premature newborn.

[unreadable] DESCRIPTION (provided by applicant): Our hypothesis is that lymphocytes, PMNs and endothelial cells exhibit a "proteolytic thermostat" which, in part, regulates the expression of cellular proteolytic activity via a series of diverse, complex sensing mechanisms comprised, in part, of selected MMP tethering molecules including MT1-MMP and CD44 and selected adhesion molecules including the Ig family member PECAM-1. In addition we postulate that these regulatory systems affect the functions of these cells at sites of inflammation. The goals of this project are to: [unreadable] [unreadable] 1. Characterize the signaling cascade(s) involved in the CD31 (PECAM-1)-mediated modulation of the induction of MMPs (MMP-2, MMP-14 [MT1-MMP] and MMP-9) in T lymphocytes and endothelial cells. We will determine the signaling cascade(s) initiated following PECAM-1 homophilic and heterophilic engagement. We will use WT and PECAM-1 null cells, PECAM-1 cytoplasmic domain truncation and exon deletion mutants and selected Y to F and S to C and S to D mutants known to affect PECAM-1-adaptor/ signaling molecule interactions. We will determine the levels of selected transcription factors known to be involved in modulating these MMPs. [unreadable] [unreadable] 2. Elucidate the roles of cell surface tethering molecules (MT1-MMP and CD44) as modulators of MMP-2 and MMP-9 expression. We will determine the signaling pathways following tethering of MMP-2 to the MT1- MMP complex and MMP-9 to CD44. We will determine MMP-2 and MMP-9 expression and activity levels in WT, MMP-2 null, MMP-9 null, MT1-MMP & null and CD44 null mice and endothelial cells and splenocytes isolated from these animals. Analyses will be performed on endothelial cells and lymphocytes following transfection with full-length, truncated and site-mutagenized MT1-MMP and CD 44 constructs. Signaling pathways will be elucidated using standard pharmacological and genetic approaches. We will determine the levels of selected transcription factors known to be involved in modulating these MMPs. [unreadable] [unreadable] 3. Elucidate the role(s) of MMPs as modulators of selected chemokine activities in experimental autoimmune encephalomyelitis (EAE). We will assess the abilities of MMP-2, -9 and 14 to proteolyze selected chemokines during the development of EAE in vivo and in lymphocyte and endothelial cell cultures. [unreadable] [unreadable] 4. We will test conclusions drawn from our in vitro data in our in vivo murine model of antigen-specific inflammation in, EAE. Specifically we will use WT, CD31 KO, MMP-2 KO, MMP-9 KO, MMP-14 and -/- and CD44 KO mice, and bone marrow chimeric mice generated from these mice. [unreadable] [unreadable] [unreadable]

Our data support the concept of variable responses to brain injury in the preterm infant populafion. We hypothesize that survival and recovery from such injuries is due in part to a coupled neurogenic/vasculoangio-genic response in the neurovascular niche areas of the brain (including the subventricular zone - SVZ) and that HIF-la induced BDNF upregulafion in the vascular system is required for endothelial cell survival, proliferation and vasculogenesis and neural progenitor cell survival, proliferafion and behavioral recovery. A systemafic in vivo and in vitro investigafion of neural stem/progenitor cells (NSCs) and their associated microvasculature in the neurovascular niche areas ufilizing a broad range of techniques is warranted in order to develop rational therapeutic approaches that opfimize recovery. Using murine models of chronic sublethal hypoxia we determined that the survival and recovery of this insult (which mimics the chronic sublethal hypoxia of the preterm infant population) is variable and depends in part upon the responsiveness of the NSCs and microvascular endothelial cells (ECs) in the neurogenic zones. We hypothesize that the upregulation of BDNF in the vascular system may be crucial for the beneficial effect of sensorimoror enrichment in these processes. This project will define the extent to which the HIF-la in the vascular endothelium is responsible for endothelial cell survival, proliferation and vasculogenesis and contributes to neural progenitor cell survival, proliferafion and behavioral recovery. This will be tested by using endothelial HIF-la deficient mice that will be exposed to hypoxia followed by standard or enriched environments. Survival, proliferation differenfiation, and apoptosis of the ECs and NSCs resident in the SVZ will be invesfigated in vivo and in vitro using 2- &3-dimensional co-cultures. The effects of NPC TrkB paracrine signaling on EC behavior will be assessed in both in vivo and in vitro assays as will white matter angiogenesis and vascular density in hEGFR over-expressing mice and HIF-la modulafion of UCP2 expression, funcfion and mitochondrion number in collaborafion with the other projects in this P01

DESCRIPTION (provided by applicant): Our hypothesis is that lymphocytes, PMNs and endothelial cells exhibit a "proteolytic thermostat" which, in part, regulates the expression of cellular proteolytic activity via a series of diverse, complex sensing mechanisms comprised, in part, of selected MMP tethering molecules including MT1-MMP and CD44 and selected adhesion molecules including the Ig family member PECAM-1. In addition we postulate that these regulatory systems affect the functions of these cells at sites of inflammation. The goals of this project are to: 1. Characterize the signaling cascade(s) involved in the CD31 (PECAM-1)-mediated modulation of the induction of MMPs (MMP-2, MMP-14 [MT1-MMP] and MMP-9) in T lymphocytes and endothelial cells. We will determine the signaling cascade(s) initiated following PECAM-1 homophilic and heterophilic engagement. We will use WT and PECAM-1 null cells, PECAM-1 cytoplasmic domain truncation and exon deletion mutants and selected Y to F and S to C and S to D mutants known to affect PECAM-1-adaptor/ signaling molecule interactions. We will determine the levels of selected transcription factors known to be involved in modulating these MMPs. 2. Elucidate the roles of cell surface tethering molecules (MT1-MMP and CD44) as modulators of MMP-2 and MMP-9 expression. We will determine the signaling pathways following tethering of MMP-2 to the MT1- MMP complex and MMP-9 to CD44. We will determine MMP-2 and MMP-9 expression and activity levels in WT, MMP-2 null, MMP-9 null, MT1-MMP &null and CD44 null mice and endothelial cells and splenocytes isolated from these animals. Analyses will be performed on endothelial cells and lymphocytes following transfection with full-length, truncated and site-mutagenized MT1-MMP and CD 44 constructs. Signaling pathways will be elucidated using standard pharmacological and genetic approaches. We will determine the levels of selected transcription factors known to be involved in modulating these MMPs. 3. Elucidate the role(s) of MMPs as modulators of selected chemokine activities in experimental autoimmune encephalomyelitis (EAE). We will assess the abilities of MMP-2, -9 and 14 to proteolyze selected chemokines during the development of EAE in vivo and in lymphocyte and endothelial cell cultures. 4. We will test conclusions drawn from our in vitro data in our in vivo murine model of antigen-specific inflammation in, EAE. Specifically we will use WT, CD31 KO, MMP-2 KO, MMP-9 KO, MMP-14 and -/- and CD44 KO mice, and bone marrow chimeric mice generated from these mice.