1982 — 1985 |
Fujiwara, Keigi |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Morphological Analysis of Contractile Systems |
0.957 |
1985 |
Fujiwara, Keigi |
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. |
Cytoskeletal Organization in Vascular Endothelium @ Harvard University (Medical School)
Stress fibers (actin filament bundles which are associated with other contractile proteins and proteins believed to be important for the control of cellular contractile activities) are well-developed in the cytoplasm of most tissue culture cells. The majority of cells in the body, however, do not have stress fibers. Among the mammalian tissue cells studied so far, only endothelial cells of the cardiovascular system contain stress fibers. Studies proposed in this application are aimed at elucidating the biological function(s) of stress fibers in vascular endothelial cells. The stress fiber of endothelial cells will be characterized by electron microscopy in order to obtain its detailed ultrastructural features, and by immunofluorescence microscopy in order to determine its macromolecular composition. Since in situ endothelial cells are difficult to manipulate experimentally and since cultured vascular endothelial cells are not physiologically identical to those of the in situ endothelium short-term organ cultures of the rat arterial wall will be used to study the stress fibers' function in situ. Stress fibers in tissue culture cells appear to play a role in cell adhesion, thus their involvement in cell adhesion will be investigated in situ. Since cell adhesion results from the interaction between the cell membrane, to which the stress fiber is anchored, and the extracellular matrix, attempts will be made to obtain evidence for a specific interaction between the stress fiber and the extracellular matrix. The long-term objective of this project is to understand how vascular endothelial cells are anchored to the basement membrane. Endothelial cells aer exposed constantly to fluid shear stress, thus tight adhesion to the basement membrane must be a major concern for this cell type. If endothelial cells lose their adhesion, serious pathological conditions such as atherosclerosis result. The investigation on the mechanism of the tight adhesion between endothelial cells and the basement membrane is an important first step toward finding a cure for these diseases.
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0.942 |
2002 — 2005 |
Fujiwara, Keigi |
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. |
Cell Adhesion Molecules in Endothelial Mechanosignaling @ University of Rochester
DESCRIPTION (provided by the applicant): Blood vessels were once thought to be an infrastructure of the body that had no significantly active function, and their biology was of little concern for biomedical investigators and physicians. However, recent progress in vascular biology has revealed that blood vessels, especially endothelial cells are metabolically highly active and perform multifaceted functions, such as synthesizing a host of physiologically active substances, receiving and transmitting signals, and controlling the passage of molecules and cells across the vessel wall. Thus, maintaining healthy endothelium should be a matter of high priority in nation's health program. There are known risk factors (such as high levels of lipids in blood, hypertension, stress, smoking, male hormone, age and genetic make-up of an individual) for atherosclerosis, and, most of them are systemic factors. However, the initial occurrence of atherosclerotic legions is limited to specific regions within the artery, suggesting that in addition to these systemic factors, some important local factor(s) must exist. The areas within the artery where atherogenesis occurs are the regions of disturbed laminar flow or decreased fluid shear stress. Many studies have shown that endothelial cell physiology is very sensitive to fluid shear stress, frictional force of flowing blood that acts on the endothelial cell surface. The project proposes to study the molecular mechanism of flow sensing by endothelial cells. The study should provide new information and insights regarding two important questions in endothelial and vascular cell biology: 1) the molecular mechanism of FSS sensing and 2) the subsequent signal transduction events. At the clinical level, our study should provide a set of new points of pharmacological and other means of intervention in order to prevent and control the development of vascular diseases, in particular atherogenesis
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1 |
2005 — 2009 |
Fujiwara, Keigi |
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--Imaging @ University of Rochester
At present, the Imaging Core of the Center for Cardiovascular Research houses one Olympus laser scanning confocal microscope (Fluoview FV300) equipped with Krypton, Argon and He/Ne lasers for confocal microscopy and Mercury and Halogen lamps for epifluorescence and DIC/phase contrast/bright field microscopy, respectively. The confocal unit is mounted on an inverted Olympus microscope (1X70) with a universal high NA condenser. For capturing non-confocal images, a Spot CCD camera is used. This confocal microscope will be used for the proposed PPG projects. There is an up-right Olympus microscope (BX51) for epifluorescence, phase contrast, and DIC observations. The scope is also equipped with a Spot CCD camera. This scope is used for routine immunofluorescence observations, especially for en face analyses of immunostained endothelial tissue. Specimens for immunofluorescence microscopy are prepared by individual investigators. Technical suggestions and advice are given to them, such as fixation and staining conditions, blocking, and mounting. Data acquisition is done with the help of the Core Director and an assistant using a TV monitor. Those who wish to use the scopes by themselves are given instructions by the Core Director. MCID Elite 6.0 (Imaging Research Inc.) is available for the studies proposed in this application. The software is capable of performing various forms of image analyses such as image enhancement, image processing and editing, and 3D reconstruction. It has also functions that are quantitative such as profiling (vertical, horizontal, rotatable, and user-traced lane definition), stereology for unbiased morphological measurements and quantification, and grain counting. The grain counting mode is applicable to cells, grains, organelles, and other discrete objects. Valid targets are discriminated from background based on their intensity, color and spatial criteria.
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1 |
2010 — 2013 |
Abe, Jun-Ichi Berk, Bradford C [⬀] Fujiwara, Keigi |
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. |
Fluid Shear Stress Signal Transduction in Endothelium @ University of Rochester
DESCRIPTION (provided by applicant): Inflammation contributes to development of atherosclerosis. Atherosclerosis is decreased in regions of steady flow associated with high shear stress (termed s-flow), compared to regions of disturbed and low flow (termed d-flow). This finding has yielded the concept that s-flow is atheroprotective and d-flow is atheropromoting. Previously we showed that s-flow activated MEK5-ERK5 in endothelial cells (EC), and inhibited tumor necrosis factor (TNF) signaling. Using a novel MEK5 inhibitor we showed that ERK5 activation was required for s-flow- mediated inhibition of TNF signaling. Conceptually there are two ways to improve EC dysfunction. The most common approach has been to activate atheroprotective mechanisms such as increasing systemic nitric oxide. However, we believe that a more elegant approach is to inhibit the atheroprone mechanisms that occur uniquely in areas of d-flow. Several findings indicate that PKCzeta-dependent signaling represents a unique atheropromoting mechanism. 1) PKCzeta promotes the adhesive phenotype of EC when activated by TNF, via stimulation of NF:B-dependent ICAM-1 expression. 2) PKCzeta activity is specifically increased in EC exposed to d-flow. 3) PKCzeta activity is required for TNF-mediated JNK and caspase-3 activation in EC. 4) Exciting preliminary data show that TNF and ONOO-, via activation of PKCzeta, enhance SUMOylation of the pro- apoptotic transcription factor p53. The SUMOylation pathway is analogous to that of ubiquitination, but SUMO conjugation involves a different enzymes including the protein inhibitor of activated STAT (PIASy) family. Our data show that PKCzeta activates PIASy SUMO E3 ligase activity, increases p53-SUMO, which increases p53 protein stability and enhances the apoptotic function of p53 (Figure). The major hypothesis of our proposal is that PKCzeta activation in EC at atheroprone areas inhibits ERK5- dependent transcriptional activity and stimulates p53-SUMOylation, thereby promoting EC inflammation and apoptosis. To define mechanisms that limit PKCzeta-dependent signal events we propose four aims. Aim 1. Show that PKC6 binds and phosphorylates ERK5, thereby inhibiting ERK5 transcriptional activity. Aim 2. Define the molecular mechanisms by which PKCzeta functions as an activator for p53 based on the concept that protein inhibitor of activated STAT y (PIASy;SUMO ligase) associates with PKCzeta increasing p53- SUMOylation, p53 expression and EC apoptosis. Aim 3. Characterize the effects of s-flow and d-flow on PKCzeta, ERK5, PIASy, and p53 function in vitro based on the hypothesis that d-flow stimulates PKCzeta activation, p53 nuclear export, and EC apoptosis. Aim 4. Show that PKCzeta and PIASy augment atherosclerosis in the apoE-/- mouse by inhibiting ERK5 activity and stimulating p53-SUMOylation. These studies should provide insight on how flow inhibits vascular inflammation and facilitate development of new therapeutic approaches to limit atherosclerosis. PUBLIC HEALTH RELEVANCE: Strokes and heart attacks are the leading cause of death in the US. Interventions such as bypass surgery and angioplasty treat acute events, but there are limited therapies to prevent the underlying disease termed atherosclerosis. We have found that an enzyme - protein kinase C-zeta - is powerfully activated in blood vessels at sites where atherosclerotic plaques develop. Here we will focus on novel approaches to inhibit the function of this enzyme. Elucidating the specific pathways by which protein kinase C-zeta modulates vessel function would provide the basis to develop new therapies to prevent atherosclerosis.
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1 |
2010 — 2013 |
Abe, Jun-Ichi Fujiwara, Keigi |
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. |
P90rsk: a Flow Responsive Mediator of Inflammation @ University of Rochester
DESCRIPTION (provided by applicant): The role of inflammation in cardiovascular disease and diabetes (DM) has become increasingly evident. Especially, a combination of risk factors such as hypertension, obesity, DM, smoking, hyperlipidemia, and genetic predisposition create a proinflammatory environment that leads to endothelial (EC) dysfunction. This dysfunction is exacerbated by disturbed blood flow (d-flow) against the EC. In the physiological state, normal EC function is maintained with the release of anti-atherosclerotic signals stimulated by laminar/steady blood flow and high shear stress (s-flow). Our data show that p90RSK activation inhibits Sentrin/SUMO- specific proteases 2 (SENP2) de-SUMOylation activity and increases both p53 and ERK5-SUMOylation, which increases p53 nuclear export and enhances the apoptotic function of p53 and inhibits ERK5-transcriptional activity and its anti-inflammatory responses. The major hypothesis is that p90RSK activation in EC at atheroprone areas inhibits ERK5-dependent transcriptional activity and stimulates p53-SUMOylation thereby promoting EC inflammation and apoptosis, especially in DM. The experimental approach will be to define the mechanisms by which p90RSK regulates ERK5, p53, EC inflammation and apoptosis in Aims 1 and 2. In Aim 3 we will use mutants and inhibitory fragments generated in aims 1 and 2 in well-defined flow environments to prove that we can mitigate the harmful effects of d-flow by inhibiting p90RSK- and SENP2-mediated inflammation and apoptosis. In Aim 4 we will use genetic mouse models to evaluate the relative roles of p90RSK and SENP2 activity in atherosclerosis. We anticipate that specific d-flow and DM-dependent p90RSK activation and subsequent ERK5 and p53-SUMOylation make EC atheroprone. The proposed studies should provide significant new information regarding two important questions in d-flow and DM-related EC dysfunction: 1. The role of p90RSK activation on SENP2 de-sumoylation activity and subsequent EC apoptosis and inflammation, and 2. The role of p90RSK-mediated ERK5 phosphorylation on EC inflammation. The concept of p90RSK-SENP2 and p90RSK-ERK5 compartmentalization (nucleus vs cytosol) in atherosclerosis is novel and highlights the importance of post-translational mechanisms in disease pathogenesis. The proteins in these pathways should be attractive drug targets since they have unique features that distinguish them from other MAPK and signal events. We believe that our novel small molecule, specific p90RSK inhibitor, should provide a new therapeutic strategy for reducing atherosclerosis in DM. PUBLIC HEALTH RELEVANCE: The role of inflammation in cardiovascular disease and diabetes has become increasingly evident. At the basic science level understanding the specific signaling events involved in these mechanisms is a key issue that will be addressed here by biochemistry, cell biology, and in vivo transgenic mice. These studies should provide insight into mechanisms by which disturbed flow promotes vascular inflammation and facilitate development of new therapeutic approaches to limit atherosclerosis, especially in DM.
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