Elaine F Reed
University Of California Los Angeles
Project start date: 1999-01-01
Project end date: 2014-12-31
Sponsored Links Excellgen http://Excellgen.com
ANTIMHC ANTIBODIY EFFECTS ON ENDOTHELIUM AND MUSCLE
Elaine F Reed, Professor
Pathologyuniversity Of California Los Angeles
office Of Research Administration
los Angeles, Ca 90095
Grant 5R01AI042819-04 from National Institute Of Allergy And Infectious Diseases IRG: SAT
Abstract: adapted from ´s ) Accelerated graft atherosclerosis, a manifestation of chronic rejection, is the leading cause of late graft failure in patients surviving the first year post transplantation. The development of antibodies to donor HLA antigens is a major risk factor associated with transplant atherosclerosis. The overal goal of this proposal is to determine the mechanism(s) through which anti-HLA antibodies stimulate endothelial cell (EC) and smooth muscle cell (SMC) activation and proliferation. In specific aim 1, the investigators will establish whether exposure of endothelial cells to anti-HLA antibodies from patients with transplant atherosclerosis is accompanied by the production of growth factors. Donor EC will be treated with recipient anti-HLA antibodies and the production of basic fibroblast growth factor (bFGF) will be assessed. The ability of anti- HLA antibodies to upregulate growth factor ligand binding will be evaluated using bFGF radioligand binding assays. The investigators will also determine whether exposure of endothelial cells to anti-HLA antibodies leads to increased permeability of the EC monolayer and de novo expression of adhesion molecules. The interaction of anti-HLA antibodies with the endothelium in the presence of cytokines will be examined. In specific aim 2, the investigators will determine whether anti-HLA antibodies stimulate the proliferation of primary cultures of donor SMC. These experiments will assess whether anti-HLA antibodies induce EC to secrete growth factors that stimulate proliferation of SMC and second whether anti-HLA antibodies bind to and stimulate SMC proliferation directly. The ability of inflammatory cytokines to augment proliferative effects of anti-HLA antibodies on SMC will be evaluated
Keywords: MHC class I antigen, antibody, atherosclerosis, disease /disorder proneness /risk, fibroblast growth factor, heart transplantation, homologous transplantation, kidney transplantation, postoperative complication, transplant rejection, vascular endothelium, vascular smooth muscle biological signal transduction, cell adhesion molecule, cell proliferation, coronary artery, cytokine, growth factor receptor, intermolecular interaction, receptor expression flow cytometry, human tissue, monoclonal antibody, polymerase chain reaction, tissue /cell culture
Project start date: 1999-01-01
Project end date: 2002-12-31
5R01AI042819-04 (2001): $182024
5R01AI042819-03 (2000): $176724
Antimhc Antibody Effects On Endothelium And Muscle
Elaine F Reed, Professor
Pathology And Laboratory Medicineuniversity Of California Los Angeles
office Of Research Administration
los Angeles, Ca 90095
Grant 5R01AI042819-09 from National Institute Of Allergy And Infectious Diseases IRG: SAT
Keywords: antibody, muscle, phosphorylation actin, adhesion, arteriosclerosis, base, biopsy, cell, cell proliferation, culture, cytoskeleton, fibroblast growth factor, flow cytometry, focal adhesion kinase, growth factor receptor, histocompatibility antigen, homologous transplantation, human, intracellular, lead, microscopy, muscle cell, paxillin, play, protein, receptor, receptor expression, role, smooth muscle, success, transplant rejection, tyrosine clinical research
Project start date: 1999-01-01
Project end date: 2008-12-31
5R01AI042819-09 (2007): $361494
5R01AI042819-08 (2006): $372291
5R01AI042819-07 (2005): $381250
5R01AI042819-06 (2004): $381250
Grants awarded to Elaine F Reed
ANTI-MHC EFFECTS ON ENDOTHELIUM
Elaine F Reed, Professor
University Of California Los Angeles, Office Of Research Administration, Los Angeles, Ca 90095
Grant 2R01AI042819-10A2 from National Institute Of Allergy And Infectious Diseases
Abstract: Transplant recipients developing anti-donor HLA antibodies after transplantation demonstrate increased risk of antibody-mediated rejection, transplant associated vasculopathies and graft loss. HLA class I signaling pathways have been implicated in these processes because ligation of class I molecules by anti-HLA antibodies initiates intracellular signals in endothelial cells that synergize with growth factor receptors to elicit survival and proliferation. We found that anti-HLA antibodies elicit different signal transduction outcomes, survival vs. proliferation, depending upon their specificity, titer and the degree of HLA antigen expression on the endothelial cell. These studies provide a paradigm for further elucidating the molecular mechanisms underlying how the concentration of antibody mediates survival and proliferation events in endothelium and their relationship to transplant outcome. Activation of anti-apoptotic and cell survival machinery in endothelial cells is augmented when cells are exposed to low concentrations of anti-HLA antibodies. In contrast, treatment of endothelial cells with high concentrations of HLA antibodies stimulates cell proliferation. This suggests that low levels of antibody binding to HLA may be beneficial to transplant survival by activating survival pathways that promote graft accommodation. On the other hand, high levels of antibody binding may have a detrimental effect on graft survival by upregulating FGFR expression, stimulating cell proliferation and increasing risk for development of transplant vasculopathy. We believe these findings are clinically relevant and may explain differences in transplant outcome in recipients producing anti-donor HLA antibodies. The overall goals of this proposal are to elucidate whether the intracellular signaling events initiated by antibody ligation of class I molecules are influenced by the specificity and concentration of the antibody and to determine the clinical relevance of class I signaling pathways in transplantation. Aim 1 will focus on HLA interactions with 2 integrins and survival and proliferation phosphorylation cascades induced by different titers of anti-HLA antibody on primary human endothelial cells. These studies will permit us to dissect the class I signaling pathways and explore the cause-effect relationships between proteins in the pathway. Aim 2 will test whether the antibody induced phosphorylation cascades identified in cultured EC are also operational in vivo. We will study the effect of different titers and specificities of anti-MHC antibody on induction of cell survival and proliferation signal transduction pathways in a RAG1 knock out murine heterotopic cardiac transplant model. Aim 3 will explore whether anti-MHC class I induced phosphorylation of proteins in cardiac transplant biopsies correlate with the presence and titer of circulating anti-donor-HLA antibodies, diagnosis of antibody-mediated acute and chronic rejection and transplant outcome. These experiments will confirm the biological relevance of class I mediated signaling in human transplantation. Studying anti-HLA antibody mediated signal transduction in the proposed in vitro and in vivo models will permit us to establish the importance of the class I signaling pathways in solid organ transplant outcome. These studies will identify key signaling proteins mediating acute and chronic antibody-mediated rejection and may permit the development of new treatment strategies. Using samples from transplant recipients, this study will develop and test class I antibody mediated signal transduction as reliable indicators of transplant outcome and provide insight into mechanisms underlying antibody mediated graft injury
Keywords: 1-Phosphatidylinositol 3-Kinase; ATP[{..}]1-phosphatidyl-1D-myo-inositol 3-phosphotransferase; Acute; Address; Antibodies; Antibody Formation; Antibody Production; Antibody Response; Apoptotic; Binding; Binding (Molecular Function); Biological; Biopsy; Cardiac; Cell Communication and Signaling; Cell Growth in Number; Cell Multiplication; Cell Proliferation; Cell Signaling; Cell Survival; Cell Viability; Cells; Cellular Proliferation; Chronic; Clinical, Transplantation, Organ; Closure by Ligation; Complement; Complement Proteins; Complex; Data; Development; Diagnosis; Dose; EC 2.7; Endothelial Cells; Endothelium; Event; Extracellular Matrix, Integrins; FAK; FAK1; FGF-R; FGFR; FK506 Binding Protein 12-Rapamycin Associated Protein 1; FK506 Binding Protein 12-Rapamycin Associated Protein 2; FK506 binding protein 12-rapamycin associated protein 1, human; FKBP-Rapamycin Associated Protein; FKBP-rapamycin associated protein, human; FKBP12 Rapamycin Complex Associated Protein 1; FRAP1 protein, human; Fibroblast Growth Factor Receptor Family; Fibroblast Growth Factor Receptors; Focal Adhesion Kinase 1; Funding; Genes, Class I; Genes, MHC Class I; Goals; Graft Rejection; Graft Survival; Grafting Procedure; Grant; Growth Factor Receptors; HL-A Antigens; HLA Antigens; Hand; Heart; Human; Human Leukocyte Antigens; Human, General; In Vitro; Injury; Integrins; Intracellular Communication and Signaling; Kinases; Knock-out; Knockout; Leiomyocyte; Leukocyte Antigens; Ligation; MHC Class I; MHC Class I Genes; Mammals, Mice; Man (Taxonomy); Man, Modern; Mediating; Mice; Modeling; Molecular; Molecular Interaction; Murine; Mus; Muscle, Involuntary; Muscle, Smooth; Myocytes, Smooth Muscle; Organ Transplantation; Organ Transplants; Organ Transplants, Including Bone Marrow for DCT; Outcome; PI-3 Kinase; PI-3K; PI3-Kinase; PTK2; PTK2 Protein Tyrosine Kinase 2; Pathway interactions; Phosphatidylinositol 3-Kinase; Phosphatidylinositol-3-OH Kinase; Phosphoinositide 3-Hydroxykinase; Phosphorylation; Phosphotransferases; Process; Progress Reports; Protein Phosphorylation; Proteins; Proto-Oncogene, Growth Factor Receptor; PtdIns 3-Kinase; Public Health; Publishing; RAFT1 protein, human; RAPT1 protein, human; Rapamycin Target Protein; Receptors, FGF; Reports, Progress; Ribosomal Protein S6; Ribosomal Protein S6 Kinase; Risk; Risk Factors; Role; S6 Kinase; S6-H4 Kinase; Sampling; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Signal Transduction Systems; Signaling; Signaling Protein; Site; Smooth Muscle Cells; Smooth Muscle Myocytes; Smooth Muscle Tissue Cell; Smooth muscle (tissue); Solid; Specificity; Testing; Time; Transphosphorylases; Transplant Recipients; Transplant Rejection; Transplantation; Transplantation Rejection; Transplantation Surgery; Type I Phosphatidylinositol Kinase; Type III Phosphoinositide 3-Kinase; Up-Regulation; Up-Regulation (Physiology); Upregulation; Vascular Diseases; Vascular Disorder; antibody biosynthesis; biological signal transduction; blood vessel disorder; cardiac allograft; clinical relevance; clinically relevant; endogenous substrate pp120; experiment; experimental research; experimental study; focal adhesion kinase; focal adhesion protein tyrosine kinase; focal adhesion-associated protein tyrosine kinase pp125FAK; gene product; heart allograft; human FRAP1 protein; immunoglobulin biosynthesis; in vivo; in vivo Model; insight; mTOR; mutant; organ allograft; organ graft; organ xenograft; p125(FAK); p125FAK; pathway; paxillin; pp125(FAK); pp125FAK; public health medicine (field); public health relevance; rapamycin and FKBP12 target 1 protein, human; receptor expression; research study; social role; transplant; transplant associated vasculopathy; transplant patient; treatment strategy
Relevance: Relevance to Public Health Studying anti-HLA antibody mediated signal transduction in the proposed in vitro and in vivo models will permit us to establish the importance of the class I signaling pathways in solid organ transplant outcome. These studies will identify key signaling proteins mediating acute and chronic antibody-mediated rejection and may permit the development of new treatment strategies. Using samples from transplant recipients, this study will develop and test class I antibody mediated signal transduction as reliable indicators of transplant outcome and provide insight into mechanisms underlying antibody mediated graft injury
Project start date: 1999-01-01
Project end date: 2014-12-31
Budget start date: 1-JAN-2010
Budget end date: 31-DEC-2010
PFA/PA: PA-07-070
2R01AI042819-10A2 (2010): $385000
INFLUENCE OF HLA ON ANTIBODIES AND TCR V GENE EXPRESSION
Elaine F Reed, Professor
Pathologycolumbia Univ New York Morningside
research Administration
new York, Ny 100277003
Grant 5R01AI033699-02 from National Institute Of Allergy And Infectious Diseases IRG: SAT
Abstract: The major threat to long term survival of allografts is chronic humoral rejection. In both cardiac and renal transplantation, chronic rejection appears to be induced by HLA antigens since the development of antibodies to donor HLA antigens is associated with graft failure. The recipient´s HLA class II genotype may play a major role in the initiation of chronic allograft rejection since class II MHC molecules differ in their ability to bind different antigenic peptides and therefore to stimulate specific helper T cells. In addition, MHC molecules may also play a role in MHC linked immune responsiveness by shaping the repertoire of antigen- responsive T cells through positive and negative selection in the thymus. The overall goal of this proposal is to study the influence of HLA molecules on the alloimmune response. In Specific Aim 1, the possibility that particular MHC class II genes are associated with responsiveness or nonresponsiveness to alloantigens will be explored. The transplant patient´s HLA class II genotype will be established using molecular typing methods. Correlation between the presence or absence of a particular HLA allele and recipient race, production of anti-HLA antibodies, number of rejection episodes and graft survival will be determined. This study will permit the identification of HLA class II alleles associated with immune responsiveness to the graft. Specific Aim 2, is to determine whether HLA class I and II molecules influence the selection of the peripheral TCR repertoire within the CD8+ and CD4+ compartments. The peripheral repertoire of CD4+ and CD8+ TCR Vbeta genes will be determined by quantitative PCR and flow cytometric methods in HLA-identical siblings and in parents and siblings who differ by one and/or two haplotypes. If a similar frequency of TCR Vbeta genes in HLA- identical siblings compared to one or two haplotype mismatched siblings is found, this would indicate that MHC guides the selection of TCR V genes. The influence of class I and class II genes in shaping the CD4 and CD8 TCR repertoire will be analyzed in HLA-B/DR cross-over families. The possible relationship between the individual´s HLA-DR genotype and the frequency of particular Vbeta genes within the CD4 compartment will be explored in families with intercrosses for particular HLA-DRB alleles
Keywords: T cell receptor, allele, gene expression, histocompatibility antigen, histocompatibility gene, transplant rejection CD4 molecule, CD8 molecule, MHC class I antigen, MHC class II antigen, antiantibody, gene frequency, genetic polymorphism, heart transplantation, histocompatibility, humoral immunity, kidney transplantation, protein sequence, sibling, twin /multiplet computer simulation, flow cytometry, histocompatibility typing, human subject, polymerase chain reaction
Project start date: 1993-09-30
Project end date: 1996-06-30
5R01AI033699-02 (1994): $150298
1R01AI033699-01A1 (1993): $150300
ANTI-HLA ANTIBODY ACTIVATION OF ENDOTHELIAL CELLS IN CARDIAC TRANSPLANTATION
Elaine F Reed, Professor
University Of California Los Angeles, Office Of Research Administration, Los Angeles, Ca 90095
Grant 5R01HL090995-03 from National Heart, Lung, And Blood Institute
Abstract: Cardiac transplant recipients exhibiting a humoral immune response to the allograft demonstrate increased risk of antibody-mediated rejection and transplant arteriosclerosis. HLA class I and class II signaling pathways have been implicated in this process because ligation of class I and class II molecules by anti-HLA antibodies initiates intracellular signals in endothelial cells that synergize with growth factor receptors to elicit cell survival and cell proliferation. Activation of anti-apoptotic and cell survival machinery in endothelial cells is augmented when cells are exposed to low concentrations of anti-HLA antibodies. In contrast, treatment of endothelial cells with high concentrations of anti-HLA antibodies stimulates cell proliferation. This suggests that lower levels of anti-HLA antibody binding to HLA may be beneficial to graft survival by activating survival signaling pathways that promote graft accommodation. On the other hand, high levels of anti-HLA antibody binding may have a detrimental effect on graft survival by upregulating FGFR expression, stimulating cell proliferation and increasing risk for development of transplant arteriosclerosis. We believe these findings are clinically relevant and may explain differences in transplant outcome in cardiac transplant patients producing anti-donor HLA antibodies. The overall goals of this proposal are to elucidate whether the intracellular signaling events initiated by antibody ligation of class I and class II molecules are influenced by the specificity and concentration of the HLA antibody and to determine the clinical relevance of class I and II signaling pathways in cardiac transplantation. Patient samples to be used in the proposed research will be obtained from the "CTOT-05 Observational Study of Alloimmunity in Cardiac Transplant Recipients". The CTOT-05 study is a prospective, non-randomized, multicenter, observational clinical trial in cardiac transplant recipients. Under Aim 1, we will characterize the ability of anti-HLA class I and class II antibodies in sera from heart allograft recipients to mediate proliferative and/or cell survival signals in endothelial cells. The IgG fraction of the serum will be tested for its ability to transduce signals via MHC class I and class II molecules in donor and surrogate endothelial cells bearing the relevant HLA antigen(s). Under Aim 2 we will assess the expression of anti-HLA antibody induced protein phosphorylation, fibroblast growth factor receptors and anti-apoptotic proteins in clinical biopsy specimens from cardiac allografts with and without anti-HLA antibody production. Understanding the class I and class II signaling pathways in the context of antibody mediated rejection and transplant arteriosclerosis is of importance for the development of new diagnostic tools and therapeutic strategies to improve patient management and cardiac transplantation outcome. Relevance to Public Health Studying anti-HLA antibody mediated signal transduction in samples from the CTOT-05 clinical trial will permit us to establish the importance of the class I and class II signaling pathways in cardiac transplant outcome. These studies will identify key signaling proteins mediating acute and chronic antibody-mediated rejection and may permit the development of new treatment strategies. This study will develop and test class I and class II antibody mediated signal transduction as reliable indicators of transplant outcome and provide insight into mechanisms underlying antibody mediated graft injury. (End of )
Keywords: 1-Phosphatidylinositol 3-Kinase; AIGF factor; ATP[{..}]1-phosphatidyl-1D-myo-inositol 3-phosphotransferase; Active Follow-up; Acute; Address; African; Alloantibodies; Allogenic; Allografting; Antibodies; Antibody Activation; Antibody Formation; Antibody Production; Antibody Response; Antibody Specificity; Apoptotic; Arteriosclerosis; Asians; Assay; Binding; Binding (Molecular Function); Bioassay; Biologic Assays; Biological Assay; Biopsy; Biopsy Sample; Biopsy Specimen; Blood Serum; Calcium Phospholipid-Dependent Protein Kinase; Calcium-Activated Phospholipid-Dependent Kinase; Cardiac; Cell Communication and Signaling; Cell Growth in Number; Cell Multiplication; Cell Proliferation; Cell Signaling; Cell Survival; Cell Viability; Cells; Cellular Proliferation; Chronic; Clinical; Clinical Data; Clinical Research; Clinical Study; Clinical Trials; Clinical Trials, Unspecified; Clinical assessments; Closure by Ligation; Data; Data Banks; Data Bases; Data Set; Databank, Electronic; Databanks; Database, Electronic; Databases; Dataset; Development; Early treatment; Endothelial Cells; Endothelium; Enrollment; Ensure; European; Event; Exhibits; FAK; FAK1; FGF-2; FGF-8 protein; FGF-R; FGF2; FGF8; FGFR; FK506 Binding Protein 12-Rapamycin Associated Protein 1; FK506 Binding Protein 12-Rapamycin Associated Protein 2; FK506 binding protein 12-rapamycin associated protein 1, human; FKBP-Rapamycin Associated Protein; FKBP-rapamycin associated protein, human; FKBP12 Rapamycin Complex Associated Protein 1; FRAP1 protein, human; Fibroblast Growth Factor 2; Fibroblast Growth Factor 8; Fibroblast Growth Factor Receptor Family; Fibroblast Growth Factor Receptors; Fibroblast Growth Factor, Basic; Focal Adhesion Kinase 1; Gamma Globulin, 7S; Genes; Genes, Class I; Genes, MHC Class I; Geographic Distribution; Goals; Graft Rejection; Graft Survival; Grafting, Heart; Growth Factor Receptors; HBGF-2; HL-A Antigens; HLA Antigens; Hand; Heart Transplantation; Heparin-Binding Growth Factor 2; Heparin-Binding Growth Factor Class II; Hispanic Populations; Hispanics; Hispanics or Latinos; Human; Human Leukocyte Antigens; Human, General; IGIV; IV Immunoglobulins; IVIG; IgG; Immune Globulin, Intravenous; Immune globulin IV; Immune response; Immunoglobulin G; Immunoglobulins, Intravenous; Immunosuppression Effect; Immunosuppressions (Physiology); Immunosuppressive Effect; Incidence; Injury; Intracellular Communication and Signaling; Intravenous Antibodies; Intravenous IG; Intravenous Immunoglobulins; Investigators; Isoantibodies; Latino Population; Leiomyocyte; Leukocyte Antigens; Ligation; MHC Class I; MHC Class I Genes; Mammals, Mice; Man (Taxonomy); Man, Modern; Mediating; Mice; Molecular Interaction; Murine; Mus; Muscle, Involuntary; Muscle, Smooth; Myocytes, Smooth Muscle; Natural immunosuppression; Observational Study; Outcome; PI-3 Kinase; PI-3K; PI3-Kinase; PKC; PTK2; PTK2 Protein Tyrosine Kinase 2; Parents; Pathogenesis; Pathology; Pathway interactions; Patients; Phosphatidylinositol 3-Kinase; Phosphatidylinositol-3-OH Kinase; Phosphoinositide 3-Hydroxykinase; Phospholipid-Sensitive Calcium-Dependent Protein Kinase; Phosphorylation; Plasmapheresis; Process; Programs (PT); Programs [Publication Type]; Prostate Epithelial Cell Growth Factor; Prostatropin; Protein Kinase C; Protein Phosphorylation; Proteins; Proto-Oncogene, Growth Factor Receptor; Protocol; Protocols documentation; PtdIns 3-Kinase; Public Health; RAFT1 protein, human; RAPT1 protein, human; Randomized; Rapamycin Target Protein; Receptors, FGF; Research; Research Personnel; Researchers; Resolution; Ribosomal Protein S6; Ribosomal Protein S6 Kinase; Risk; Risk Factors; Role; S6 Kinase; S6-H4 Kinase; SCHED; Sampling; Schedule; Serum; Severities; Signal Pathway; Signal Transduction; Signal Transduction Systems; Signaling; Signaling Protein; Site; Smooth Muscle Cells; Smooth Muscle Myocytes; Smooth Muscle Tissue Cell; Smooth muscle (tissue); Spanish Origin; Specificity; Surface; Testing; Therapeutic; Therapeutic Plasma Exchange; Therapeutic Plasmapheresis; Transplant Recipients; Transplant Rejection; Transplantation; Transplantation Rejection; Transplantation, Cardiac; Type I Phosphatidylinositol Kinase; Type III Phosphoinositide 3-Kinase; United States; Up-Regulation; Up-Regulation (Physiology); Upregulation; Vascular Diseases; Vascular Disorder; Vascular Endothelial Cell; ing; allograft rejection; alloimmunity; androgen-induced growth factor; antibody biosynthesis; bFGF; base; biological signal transduction; blood vessel disorder; cardiac allograft; cardiac graft; clinical data repository; clinical data warehouse; clinical investigation; clinical relevance; clinically relevant; cohort; data repository; endogenous substrate pp120; enroll; experience; focal adhesion kinase; focal adhesion protein tyrosine kinase; focal adhesion-associated protein tyrosine kinase pp125FAK; follow-up; gene product; heart allograft; heart transplant; hispanic community; host response; human FRAP1 protein; immunoglobulin biosynthesis; immunoresponse; immunosuppression; improved; in vivo; insight; isoimmunity; mTOR; new diagnostics; next generation diagnostics; novel; novel diagnostics; oriental; p125(FAK); p125FAK; pathway; patient population; paxillin; pp125(FAK); pp125FAK; programs; prospective; public health medicine (field); randomisation; randomization; randomly assigned; rapamycin and FKBP12 target 1 protein, human; receptor expression; relational database; response; sample collection; social role; specimen collection; tool; transplant; transplant patient; treatment strategy
Project start date: 2008-05-01
Project end date: 2011-03-31
Budget start date: 1-APR-2010
Budget end date: 31-MAR-2011
PFA/PA: RFA-HL-07-009
5R01HL090995-03 (2010): $385000
ANTIMHC ANTIBODIY EFFECTS ON ENDOTHELIUM AND MUSCLE
Elaine F Reed, Professor
Pathologycolumbia University Health Sciences
columbia University Medical Center
new York, Ny 100323702
Grant 1R01AI042819-01A1 from National Institute Of Allergy And Infectious Diseases IRG: SAT
Abstract: adapted from ´s ) Accelerated graft atherosclerosis, a manifestation of chronic rejection, is the leading cause of late graft failure in patients surviving the first year post transplantation. The development of antibodies to donor HLA antigens is a major risk factor associated with transplant atherosclerosis. The overal goal of this proposal is to determine the mechanism(s) through which anti-HLA antibodies stimulate endothelial cell (EC) and smooth muscle cell (SMC) activation and proliferation. In specific aim 1, the investigators will establish whether exposure of endothelial cells to anti-HLA antibodies from patients with transplant atherosclerosis is accompanied by the production of growth factors. Donor EC will be treated with recipient anti-HLA antibodies and the production of basic fibroblast growth factor (bFGF) will be assessed. The ability of anti- HLA antibodies to upregulate growth factor ligand binding will be evaluated using bFGF radioligand binding assays. The investigators will also determine whether exposure of endothelial cells to anti-HLA antibodies leads to increased permeability of the EC monolayer and de novo expression of adhesion molecules. The interaction of anti-HLA antibodies with the endothelium in the presence of cytokines will be examined. In specific aim 2, the investigators will determine whether anti-HLA antibodies stimulate the proliferation of primary cultures of donor SMC. These experiments will assess whether anti-HLA antibodies induce EC to secrete growth factors that stimulate proliferation of SMC and second whether anti-HLA antibodies bind to and stimulate SMC proliferation directly. The ability of inflammatory cytokines to augment proliferative effects of anti-HLA antibodies on SMC will be evaluated
Keywords: MHC class I antigen, antibody, atherosclerosis, disease /disorder proneness /risk, fibroblast growth factor, heart transplantation, homologous transplantation, kidney transplantation, postoperative complication, transplant rejection, vascular endothelium, vascular smooth muscle biological signal transduction, cell adhesion molecule, cell proliferation, coronary artery, cytokine, growth factor receptor, intermolecular interaction, receptor expression flow cytometry, human tissue, monoclonal antibody, polymerase chain reaction, tissue /cell culture
Project start date: 1999-01-01
Project end date: 1999-08-31
1R01AI042819-01A1 (1999): $54469
Antimhc Antibody Effects On Endothelium And Muscle
Elaine F Reed, Professor
University Of California Los Angeles Office Of Research Administration Los Angeles, Ca 90095
Grant 2R01AI042819-05 from National Institute Of Allergy And Infectious Diseases IRG: SAT
Abstract: Accelerated transplant arteriosclerosis (TA), a manifestation of chronic rejection, is the leading cause of graft failure. The development of anti-HLA antibodies to donor HLA antigens is a major risk factor associated with TA. The overall goal of this proposal is to elucidate the signal transduction pathways that mediate HLA class I induced cell proliferation and the development of TA. Under specific aim #1, we will establish whether anti-HLA antibodies induce tyrosine phoshphorylation of focal adhesion proteins and assembly of signaling complexes via an actin cytoskeleton dependent pathway in endothelium and smooth muscle. For this, we will establish whether exposure of cultured endothelial cells (EC) and smooth muscle cells (SMC) to monoclonal anti-HLA antibodies and anti-HLA antibodies from transplant patients with chronic rejection is accompanied by FAK phosphorylation and the generation of FAK/Src/Paxillin signaling complexes. We will determine if the phosphorylation of focal adhesion proteins is accompanied by alterations in the organization of the actin cytoskeleton and in the assembly of focal adhesions. We will explore the role of ROK, MLC phosphatase and ERK as upstream components of the class I signaling pathway. We will also determine if class I signaling stimulates anti-apoptotic signals by inducing tyrosine phosphorylation of PI3-kinase, Akt and Bad. Under specific aim #2, we will identify the signaling pathways leading to MHC class I induced FGF receptor translocation in EC and SMC. For this, we will determine the role of the actin cytoskeleton, phosphorylation of FAK, Src, paxillin and assembly of focal adhesions in class I mediated FGFR translocation to distinct subcellular locations using flow cytometry and confocal microscopy. We will also assess the importance of ROK, ERK, MLC phosphatase in class I mediated FGF receptor translocation. The contribution of the FGF receptor tyrosine kinase activity and ERK phosphorylation to class I induced cell proliferation will be established. Under aim #3, we will assess the expression of class I induced tyrosine phosphorylation, FGF receptors and anti-apoptotic proteins in clinical biopsy specimens from cardiac allografts with and without evidence of transplant arteriosclerosis. We will determine the correlation between protein phosphorylation events and protein expression with the incidence and time of onset of transplant arteriosclerosis and development of anti-HLA antibodies.
Keywords: biological signal transduction, cell proliferation, smooth muscle, vascular endothelium, MHC class I antigen, antibody formation, arteriosclerosis, enzyme activity, fibroblast growth factor, focal adhesion kinase, growth factor receptor, heart transplantation, ligand, mitogen activated protein kinase, monoclonal antibody, muscle cell, paxillin, phosphatidylinositol 3 kinase, phosphorylation, receptor expression, transplant rejection, biopsy, clinical research, confocal scanning microscopy, flow cytometry, human subject, tissue /cell culture
Project start date: 1999-01-01
Project end date: 2007-12-31
2R01AI042819-05 (2003): $305000