Pre-B-cell colony enhancing factor (PBEF) is a unique, little-studied cytokine, whose relevance to the lungpathophysiology is unknown. Recent work by the PI has provided the first evidence of PBEF expression inlung tissues and overexpression in acute lung injury (ALI) as well as the association of PBEF geneticvariants with susceptibility to ALI. The haplotype weighted analysis of single nucleotide polymorphism (SNP)T-1001G and C-1543T in the PBEF gene promoter revealed a susceptible haplotype GC with a 7.7-foldhigher risk of ALI. Reporter gene assays indicated that theses variants affected transcription rate. Our recentdata suggest that PBEF affects pulmonary endothelial cell permeability in vitro and increased pulmonaryedema in C57 BL/6 mice in vivo. In addition, stealth PBEF siRNA significantly inhibited TNF-alpha mediatedincrease ofIL-8 secretion, a known edematogenic factor in ALI, in pulmonary cells. All these results supportPBEF as a potential novel candidate gene and biomarker in ALI. To further detail underlying molecularmechanisms of PBEF in the pathogenesis of ALI, we hypothesize that patients with the genetically-determined susceptible haplotype in the PBEF promoter region are more susceptible to the upregulation bymechanical forces and inflammatory stimuli, two most inciting risk factors to ALI; increased PBEF expressioncontribute to the increased susceptibility to ALI by stimulating expression of other inflammatory cytokinessuch as IL-8, which leads to the pulmonary artery endothelial and epithelial cell barrier dysfunction andincreased pulmonary permeability, resulting in the pathogenesis of ALL The goal of this application is todefine the physiologic and molecular significance of the identified PBEF polymorphisms. Furthermore, wewill determine the pathophysiological role of PBEF in inflammatory lung injury with a specific focus on a roleof PBEF in increased lung vascular leak/permeability, a pathophysiological feature of ALI, both in tissueculture system in vitro and in Pbef gene knock out C57 BL/6 mice in vivo.
Specific Aim #1 will functionallycharacterize PBEF gene promoter SNPs utilizing the reporter gene and electrophoretic mobility shift assays.
Specific Aim # 2 will examine the role of PBEF gene expression in endothelial cell barrier regulation andcontractility in vitro by evaluating endothelial cell transendothelial electric resistance, stress fiber formation,and phosphorylation of myosin light chains.
Specific Aim #3 will define the signal transduction pathwaysofPBEF-mediated endothelial barrier regulation by examining PBEF- regulated genes and interacting partners.
Specific Aim #4 will examine the in vivo role of PBEF in a murine model of ALI using Pbef knock out andover-expressing animals. Together, these novel and highly translational studies using the genomic andgenetic approaches will provide new insight into molecular mechanisms of PBEF function in ALI, which maylead to the development of novel diagnostic modalities and therapeutic strategies in ALI.PERFORMANCE SITE(S) (organization, city, state)The University of ChicagoChicago, ILPHS 398 (Rev. 09/04) Page 2 Form Page 2 Principal Investigator/Program Director (Last, First, Middle): Ye, Shui Q.KEY PERSONNEL. See instructions. Use continuation pages as needed to provide the required information in the format shown below.Start with Principal Investigator. List all other key personnel in alphabetical order, last name first.Name eRA Commons User Name Organization Role on ProjectYe, Shui Q. qshuM The University of Chicago Principal InvestigatorGarcia, Joe G.N.Adyshev, DjanybekMoitra, JaideepVerin, AlexanderWang, XiaojunZhang, Li Q.The University of Chicago Co-investigatorThe University of Chicago Research Professional (Associate)The University of Chicago Co-investigatorThe University of Chicago Co-investigatorThe University of Chicago Postdoctoral ScholarThe University of Chicago Co-investigatorOTHER SIGNIFICANT CONTRIBUTORSName Organization Role on ProjectBirukov, Konstantin The University of Chicago Resource PersonCox, Nancy The University of Chicago Resource PersonReddy, Sekhara Johns Hopkins University ConsultantWang, Yanlin Johns Hopkins University ConsultantHuman Embryonic Stem Cells S No Q YesIf the proposed project involves human embryonic stem cells, list below the registration number of the specific cell line(s) from the following list:http://stemcells.nih.qov/registrv/index.asp. Usecontinuation pages as needed.If a specific line cannot be referenced at this time, include a statement that one from the Registry will be used.Cell LineDisclosure Permission Statement. Applicable to SBIR/STTR Only. See instructions. Yes NoPHS 398 (Rev. 09/04) Page 3 Form Page 2-continuedNumber the following pages consecutivelythroughoutthe application. Do not use suffixes such as 4a, 4b. Principal Investigator/Program Director (Last, First, Middle): Ye, Shui Q.The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page. RESEARCH GRANT TABLE OF CONTENTSPage NumbersFace Page 1Description, Performance Sites, Key Personnel, Other Significant Contributors, and HumanEmbryonic Stem Cells 2-3Table of ContentsDetailed Budget for Initial Budget Period (or Modular Budget) 5-6Budget for Entire Proposed Period of Support (not applicablewith Modular Budget) N/ABudgets Pertaining to Consortium/Contractual Arrangements (not applicable with Modular Budget) N/ABiographical Sketch - Principal Investigator/Program Director (Not toexceed four pages) 7-10Other Biographical Sketches (Not to exceed four pages for each - Seeinstructions) 11-41Resources. 42Research Plan. 43-69Introduction to Revised Application (Not to exceed 3 pages) 43-45Introduction to Supplemental Application (Not to exceed one page) N/A A.
Specific Aims 4 6-47 B. Background and Significance 47-50 C. Preliminary Studies/Progress Report/ (Items A-D: not to exceed 25 pages*) 50-58 Phase I Progress Report (SBIR/STTR Phase II ONLY) * SBIR/STTR Phase I: Items A-D limited to 15 pages. D. Research Design and Methods 58-69 E. Human Subjects 69 Protection of Human Subjects (Required if Item 4 on the Face Page is marked 'Yes') N/A Inclusion of Women and Minorities (Required if Item 4 on the Face Page is marked 'Yes' and is Clinical Research).... N/A Targeted/Planned Enrollment Table (for new and continuing clinical research studies) N/A Inclusion of Children (Required if Item 4 on the Face Page is marked 'Yes') N/A Data and Safety Monitoring Plan (Required if Item 4 on the Face Page is marked 'Yes' and a Phase I, II, or III clinical trial is proposed) N/A F. Vertebrate Animals 69-70 G. Literature Cited 70-74 H. Consortium/Contractual Arrangements 74 I. Resource Sharing 74 J. Letters of Support (e.g., Consultants) 74 Commercialization Plan (SBIR/STTR Phase II and Fast-Track ONLY) N/AChecklist. 79Appendix (Five collated sets. Nopage numbering necessary for Appendix.) Check if Appendix is IncludedAppendices NOT PERMITTED for Phase I SBIR/STTR unless specifically solicitedNumber of publications and manuscripts accepted for publication (not to exceed 10) 10 Other items (list):PHS 398 (Rev. 09/04) Page 4 Form Page 3
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