Alan Fine - US grants

The goal of this research is to develop and test a general class of models for the generation of neuronal dendritic shapes; these models are biologically constrained and based on diffusion- controlled growth. It is evident that dendritic arbors gave self -similar structures over a range of scales, consistent with the concept that shape is controlled by the diffusion of chemical species. These concentration fields result from the interaction of dynamic influx, extrusion and sequestration of the growth- regulating factors. In particular, it is hypothesized that calcium is such a factor, and that the local calcium ion concentration beneath the cell membrane is the specific immediate determinant of neuronal growth. The investigators will develop biologically constrained physical models for the growth of curved membrane surfaces in the presence of these diffusive fields, and will use computational methods to explore the evolution of these models under different biologically-plausible conditions. Most importantly, they will test these models by comparing the computed morphogenesis with the development of real neurons under similar imposed conditions. This research may yield an understanding of the mechanisms controlling the development of neuronal form, which influences the electrical properties and synaptic interconnections of neurons that are essential to normal brain function.***//

CD95 molecule; ligands; cytokine; asthma; disease /disorder model; cooperative study; biological signal transduction; tissue /cell culture; genetically modified animals; laboratory mouse;

The eye is equipped with a distinct ability to restrain inflammatory activity. This unusual property of the eye is mediated, in art, by the ability of cell surface corneal derived Fas ligand (FasL) to induce apoptosis by engaging Fas on influxing inflammatory cells. Establishment of ocular inflammation, therefore, likely involves surmounting the immune regulatory effects of corneal-derived FasL. Thus, we hypothesize that alterations in the expression or function of corneal derived FasL regulates, in part, the inflammatory state of the eye. The understanding of the factors which regulate Fas dependent killing in the cornea has been limited by the lack of an adequate in vitro system which allows for the systematic analyses of this phenomenon, and y a lack of information regarding the genetic mechanisms which restrict FasL expression to the cornea. In this regard, we have established a FasL expressing corneal cell culture system in which corneal cells specifically kill T cells but not B cell Fas+ immune cell targets. Because release of the inactive soluble FasL is likely to be a key site in the regulation of Fas killing, we have also developed an ELISA assay which allows for the specific measurement of the released, cleaved soluble form in cell culture medium. Moreover, we have isolated the mouse FasL promoter so that the key DNA elements and transcription factors controlling FasL expression can be identified. Consistent with its proinflammatory actions in eye tissue, we found that the cytokine IL-1beta markedly depresses the ability of corneal cells to induce apoptosis in Fas+ targets. Furthermore, our deletion analyses and gel shift studies indicate that transcription factors which bind to a promoter segment located between -332 and -228 FasL expression in corneal cells. In this grant, we will further examine the ability of specific inflammatory mediators, including IL-I, to regulate corneal cell mediated apoptosis; mechanisms responsible for modulating FasL activity will be identified. The role of adhesion molecules in the differential capacity of primary and immortalized corneal cells to kill particular T and B cell targets will be investigated. We will also further characterize the genetic basis for FasL expression in the cornea. We believe these studies should provide novel insight into a key mechanism which normally serves to restrain ocular inflammation but may be suppressed in conditions such as corneal transplant rejection and anterior uveitis.

DESCRIPTION (provided by applicant): During lung injury, T1 cells, which represent most of the gas-exchanging surface of the lungs are preferentially injured. In the current paradigm, injury signals T2 cells, as the stem cell of the alveolus to proliferate and to differentiate into new T1 cells. The inability of this process to sufficiently replenish T1 cells is a leading factor in the mortality and morbidity of conditions such as ARDS and broncho-pulmonary dysplasia. To date, direct therapeutic delivery of replacement cells has not been feasible because of the lack of a reproducible source of alveolar epithelial progenitors. Recent work demonstrating that bone marrow cells can give rise to cell types previously held to originate from only one of the different embryonic cell layers, however, suggests the possibility of using cultured bone marrow cells as an alternative source of alveolar epithelial cell precursors. In this regard, we found that intravenously delivered plastic adherent bone marrow cells (marked with E. Coli beta-galactosidase), engrafted as clusters of cells with the morphological and molecular characteristics of T1 cells. During engraftment, we did not detect any labeled T2 cells at early and late time points after injection and found, that expression of the T1 cell specific surface marker T1a occurs after incorporation of cells into the alveolar surface. We also found that engraftment in large airway epithelium occurred after intra-tracheal injection of marrow cells. Our objective in this proposal is to extend this preliminary data by addressing fundamental questions relating to the patterning of lung engraftment, the assumption of a T1 cell phenotype, and the capacity of marrow cells to engraft as differentiated airway epithelium. To do this, we will determine the expression of specified marrow markers and lung epithelial markers as cells home to the lung and differentiate into T1 cells. This information will be correlated with marker analyses of marrow cells prior to injection to help develop strategies to enrich for cells with engraftment potential. In addition, the basis for type 1 cell cluster formation, and the role of different phases and types of acute lung injury on the pattern of lung engraftment will be determined. Lastly, we will characterize the kinetics, and we will further identify the marrow cell type that engrafts as airway epithelium. These studies, we believe, will help provide a foundation for the application of cell-based therapies for conditions associated with extensive damage to the epithelial surfaces of the lung.

DESCRIPTION (provided by applicant): This grant represents a collaborative effort of investigators with expertise in marrow stem cell isolation and alveolar epithelial biology. One objective is to explore 2 novel strategies to enhance the reconstitution of the damaged alveolar gas exchange surface with intravenously administered bone marrow cells. These studies build upon our earlier work in which we demonstrated that adherent marrow cells can serve as an alternative source of alveolar epithelial progenitors during bleomycin-induced lung injury in mice. These initial observations, though promising, constitute a proof of concept, since we observed only limited replacement of recipient alveolar gas exchange surface. To apply this type of technology as a therapy for acute alveolar injury syndromes (i.e ARDS), will require, however, extensive alveolar engraftment. For one augmentation strategy, we will evaluate 4 types of purified marrow preparations that are endowed with a greater intrinsic capacity for trans-differentiation, and tissue reconstitution. The other strategy will take advantage of the central role of chemokine signaling in the homing of circulating cells to target tissues. By engaging chemokine receptors of pertinent progenitor cells as they traverse the lung, we hope to facilitate migration of cells into the alveolar space. To do this, we will first generate a profile of chemokine receptor expression in the injected cell population. Using this as a guide, we will instillate selective chemokines intra-tracheally prior to the administration of marrow cells. In the second objective of this proposal, we will extend our preliminary data showing that resident lung cells possess phenotypes similar to marow-derived stem cells. For these studies, we will use highspeed flow cytometry to identify and isolate these cells in digested lung specimens. We will employ immuno-histochemistry and in situ hybridization to localize these cells in tissue sections. Finally, we will use defined in vitro and in vivo systems to assess the capacity of these isolated lung cells to differentiate along lung or hematopoietic cell fates.

Abstract not provided.

bioimaging /biomedical imaging; technology /technique development

[unreadable] DESCRIPTION (provided by applicant): In this revised R21 grant, we propose to further characterize a putative adult lung mesenchmyal progenitor cell. We initially isolated such cells using a high-speed cell sorting strategy taking advantage of properties that typify mesenchymal progenitors from other sites. These properties include: 1) a Sca-1+/lin neg. surface profile 2) an ability to efflux Hoechst dye (so-called SP cells), 3) negative hematopoietic marker CD45 status, and 4) expression of primitive mesenchymal genes. Additional phenotyping indicate these cells are not related to fibrocytes, but rather display features of classical multipotent marrow mesenchymal stem cells (MSCs). These shared features include expression of similar markers, a capacity to differentiate into fat, smooth muscle, or cartilage, and proliferation in an undifferentiated state under select in vitro conditions. We, thus, hypothesize that the adult lung contains a distinct mesenchymal progenitor cell that can be isolated based on dye efflux and surface phenotype. In Aim 1 we will further define the phenotype of this cell, and we will clarify whether cells are unipotent or multipotent. We also propose a set of exploratory studies to identify candiate miRNAs that maintain cells in an undifferentiate state. Using flow cytometry, we will fractionate cells based on cell size and expression of select surface antigens and then test defined fractions for the ability to differentiate into fat, smooth muscle, and cartilage. After we elucidate the mesenchymal progenitor phenotype, we will evaluate the differentiation repertoire of single cells. To do this, individual cells will be clonally expanded and tested for their ability to differentiate along different mesenchymal cell fates. In the last part of this aim, we will use miRNA expression microarrays and real-time PCR to evaluate miRNA expression patterns in undifferentiated and differentiated cells. Aim 2 is focused on examing the engraftment potential of this putative lung mesenchymal progenitor cell in the lung in vivo. To survey engraftment, we will inject GFP+ cells into 2 distinct models that involve tissue remodeling either in the proximal (CC10-IL-9 transgenic) or distal lung (bleomcyin). In this work, we will evaluate the impact of in vitro cell population expansion on engraftment. Through this work, we hope to establish a foundation for the types of studies that characterize organ systems in which stem/progenitor cell biology is more developed. [unreadable] [unreadable] [unreadable]

Budgets; Cell Isolation; Cell Segregation; Cell Separation; Cell Separation Technology; Cell/Tissue, Immunohistochemistry; Cells; Computer Programs; Computer software; Computers; Consultations; Cytofluorometry, Flow; Cytometry; DISSEC; Daily; Devices; Dissection; Educational process of instructing; Electromagnetic, Laser; Ensure; Equipment; Flow Cytofluorometries; Flow Cytometry; Flow Microfluorimetry; Fluorescence Microscopy; Gene Expression; Genetics, in situ Hybridization; Glass; Goals; Hand; Histological Technics; Histological Techniques; History; Human Resources; IHC; Image; Image Analyses; Image Analysis; Immunohistochemistry; Immunohistochemistry Staining Method; In Situ; In Situ Hybridization; Individual; Investigators; Lasers; Lung; Maintenance; Maintenances; Manpower; Methods; Methods and Techniques; Methods, Other; Microfluorometry, Flow; Microscope; Microscopy; Microscopy, Fluorescence; Microscopy, Light, Fluorescence; Moab, Clinical Treatment; Monoclonal Antibodies; Paraffin; Paraffin waxes and Hydrocarbon waxes; Performance; Phase; Postdoc; Postdoctoral Fellow; Procedures; Protocol; Protocols documentation; ROC Analysis; Radiation, Laser; Reagent; Recording of previous events; Research Associate; Research Personnel; Research Resources; Researchers; Resources; Respiratory System, Lung; Slide; Software; Speed; Speed (motion); Staining method; Stainings; Stains; Teaching; Technics, Histologic; Techniques; Techniques, Histologic; Training; base; cell sorting; computer program/software; data mining; datamining; design; designing; experience; flow cytophotometry; image evaluation; imaging; in situ Hybridization Staining Method; instrument; interest; lung development; personnel; post-doc; post-doctoral; pulmonary; success

[unreadable] DESCRIPTION (provided by applicant): In this proposal, we seek to understand the role of exogenously derived CD45+ hematopoeitic cells in the development of the lung's innate immune system, and in the maturation of the pulmonary vasculature. Notably, our data show that the embryonic lung contains 2 anatomically distinct sets of CD45+ cells. One CD45+ population localized to the mesenchyme, contains cells of early and late macrophage/myeloid lineage. This finding along with data showing that mature macrophages emerge in isolated embryonic lung cultures exposed to LPS, suggest that the embryonic lung itself is a site of myeloid differentiation. The other CD45+ population is most apparent in late fetal life (E18), aggregated around branches of the pulmonary artery. Many of these cells co-express smooth muscle actin, are flattened, and appear to be in various stages of incorporation into the vessel wall; further work suggests that notch-3 dependent signaling controls their fate. Herein, we propose the following central hypothesis: one fetal CD45+ population locally differentiates in response to mesenchyme-derived signals to help establish the lung's innate immune system, whereas the second peri-vascular CD45+ population directly contributes to maturation of the pulmonary artery wall. In Aim 1, we will focus on the CD45+ mesenchymal site. Our studies will clarify the ontogeny of myeloid cell maturation and identify lung parenchymal cells that produce signals stimulating macrophage and dendritic cell differentiation. Using lung embryonic cultures, we will then define the precise regions of macrophage and dendritic cell differentiation, and evaluate the role of the lung epithelium in these events. In Aim 2 we will focus on the peri-vascular CD45+ cells. We will further define their phenotype, evaluate their distribution in other embryonic tissues, and determine the importance of PU1 dependent myeloid cell differentiation for their development. In the final set of studies, we will focus on the role of notch-3 in cell fate determination and identify the functionally relevant ligand-notch-3 binding interaction that is operative in this cell population. Through this work, we will clarify how interactions between the developing lung and the hematopoeitic system ensure that the newly born lung is ready for air-breathing. PROJECT NARRATIVE: Currently, there is little or no information regarding how cells derived from the fetal circulation contribute to lung development. Our studies suggest that such cells are not only involved in establishment of the lung's immune system, but are also involved in development of the lung's blood vessels. This work will, thus, fill in major gaps in knowledge and should foster a new developmental paradigm for understanding how the embryonic lung becomes equipped air breathing. Elucidating these developmental processes is critical for advancing the treatment of congenital lung diseases, and the specific lung diseases that afflict premature infants. [unreadable] [unreadable] [unreadable]

base; Budgets; Cells; Consultations; data mining; Equipment; Flow Cytometry; Glass; Human Resources; Image Analysis; In Situ Hybridization; Individual; instrument; Lung; Maintenance; Methods; Microscopy; Paraffin; Performance; Procedures; Protocols documentation; Research Personnel; Slide; Staining method; Stains; Techniques; Training

DESCRIPTION (provided by applicant): Fibrocytes are thought to be a distinct marrow-derived cell that contributes to collagen accumulation during lung injury. Fibrocytes are defined as CD45+/type I Col I+, underscoring their putative, simultaneous hematopoietic and connective tissue phenotype. Limitations in methods used to identify and isolate fibrocytes along with deficiencies in understanding their basic biology, however, restrict progress in the field, and fuel skepticism regarding their role in lung fibrogenic reactions. To date, methods used to study fibrocytes have primarily provided indirect information due to the fact that fibrocyte identification and isolation techniques do not yield viable cells directly;in this regard isolation involves prolonged culturing of monocytic cells in vitro. In response, we developed a strategy to directly isolate viable fibrocytes, and are seeking R21 funding to use this technology to advance the field and establish a foundation for future work. For this, we employ a transgenic mouse that expresses GFP under control of the type I Col promoter (Col-GFP). From this mouse, CD45+/GFP+ cells can be readily isolated by flow cytometry. Of note, GFP+ cells are actively transcribing Col mRNA;thus CD45+/GFP+ cells display properties that classically define fibrocytes. In the normal and 5 d bleomycin-treated lung, fibrocytes comprise ~8-9% of total lung CD45+ cells and express myeloid markers. With injury there is a ~2-fold increase in the absolute number of lung fibrocytes accompanied by a 15-fold increase in type I Col gene mRNA. These findings raise the basic question: is the expansion and activation of fibrocytes in injury localized to resident or recruited cells? To answer this question, we also developed a thoracic shielding method that protects resident lung CD45+ cells during transplantation, and as a result, supports development of chimeras with lung CD45+ and marrow CD45+ cell populations that each expresses a different fluorescent marker. Having the shielding model and Col-GFP mice in hand provides a unique opportunity to answer this basic question, and at the same time to develop a knowledge base for lung fibrocytes whose validity is not undermined by technical concerns. We, thus, propose to provide basic information regarding their localization and ontogeny (Aim 1), to test the hypothesis that they are a stable resident population (Aim 1), to test the hypothesis that collagen activation occurs in resident fibrocytes during lung injury (Aim 2), and to generate 2 comparative genomic signatures that will provide key information about lung fibrocyte identity, new candidate markers, and regulatory pathways controlling their activation (Aim 2). The long-term objective is to lay the groundwork for the rationale design of reagents to definitively resolve fibrocyte fate and function in lung fibrosis. PUBLIC HEALTH RELEVANCE: This objective of this proposal is to identify and understand the complex molecular pathways that control how the body responds to a lung injury. It is our hope that findings from this work will facilitate the design of treatments for that will help with the healing from lung injury.

DESCRIPTION (provided by applicant): Asthma is a common disease with several effective treatments, including inhaled steroids and ¿-agonists. Despite this, a small subpopulation of patients has a severe unrelenting course associated with airway remodeling. A central feature of airway remodeling is alteration in bronchial smooth muscle (BSM) phenotype, which is classically characterized by expansion of cell number and size, and increased hyper- reactivity to specific and non-specific agonists. While fundamental to asthma pathogenesis and its clinical manifestations, a lack of knowledge regarding the basis for a deranged BSM phenotype is an ongoing, unresolved, issue in the field. This is manifested by the paucity of information regarding the molecular signals underlying bronchial hyper-reactivity and by the lack of treatments directed specifically at reversing the asthmatic BSM phenotype. One major contributing factor to this state-of-affairs is the lack of tools/methodologies that support the high fidelity isolation of pure BSM cells from asthmatic lungs for analysis. To overcome this, we developed a unique transgenic mouse in which BSM singly express a green fluorescent protein (hrGFP) whereas vascular smooth muscle express green (hrGFP) and red fluorescent proteins (dsRed); thereby providing for the first time a reliable methodology for separating each of these two smooth muscle cell populations from one another, and from other lung cells using flow cytometry. Using this unique mouse, our plan is to perform comprehensive mRNA and miRNA profiling of BSM RNA to test the following broad based hypothesis: 1) BSM express a distinct genetic signature and 2) alterations in this signature mediate asthmatic BSM phenotypes. Our plan is to use the profiling data to generate lists of complete and differentially expressed mRNAs and miRNAs in normal and asthmatic BSM. Relationships between deregulated miRNAs, mRNA expression, and the identity of active signaling pathways in asthmatic BSM will be examined by bioinformatic and functional studies. At the end of this work, we will have initiated a process to fill a marked knowledge void in the asthma field and will have established a foundation for a variety of future studies. PUBLIC HEALTH RELEVANCE: Asthma involves changes in the function and properties of the muscle that surrounds the bronchial tube. While central to the many of the symptoms associated with asthma including wheezing and shortness of breath, the molecular signals that cause changes in bronchial muscle are poorly understood. The objective of this proposal is to use several unique tools and methodologies that we developed to identify key molecular signals that underlay the change in bronchial smooth muscle in asthma.

DESCRIPTION (provided by applicant): To what extent mesothelial-derived cells contribute to lung development and post-natal repair is an open and basic question for the field. The objectives of this grant are to address this fundamental issue and to assess the key role of the Wilm's tumor 1 transcription factor (WT1) in these events. Using mouse lines that carry WT1 alleles with a knock-in Cre recombinase and GFP genes, we generated preliminary data leading to 3 hypotheses that will be examined: 1) the fetal mesothelium contains progenitors for differentiated mesenchymal lung cells 2) WT1 controls the expression of key genes, such as hedgehog (Hh) pathway constituents that control mesothelial migration into the fetal lung, and 3) mesothelium-derived cells contribute to post-natal lung repair and re-growth. To summarize, we found that WT1 is selectively expressed in the lung mesothelium from E11.5 to E16 and is undetectable in the adult. We identified a similar temporal pattern of WT1 expression in the primate lung, suggesting a conserved mesothelial WT1 program across mammalian species. Lineage tracing showed that mesothelium-derived cells give rise to a substantial number of bronchial smooth muscle cells (BSM), along with other parenchymal lung cells whose identities will be established (Aim 1). We observed that WT1 expression coincides with mesothelial cell entry into the underlying lung and active Hh signaling. Mechanistically, we found that WT1 binds to the promoters of multiple Hh pathway genes in mesothelial cells, and that selective loss of mesothelial Hh signaling markedly attenuates entry into the underlying lung in association with diminished expression of EMT genes. These data point to a key role for WT1 in the fetal mesothelium, controlling pathways such as Hh signaling that are involved in migration and EMT, which will be further explored (Aim 2). Interestingly, preliminary data indicate that WT1 is reactivated during lung re-growth post-pneumonectomy whereas WT1 is not re-activated in inflammatory lung injuries, such as asthma and fibrosis. These findings suggest 2 models for how the mesothelium may contribute to lung remodeling in post-natal life. In model 1, the fetal WT1-regulated mesothelial program is re-activated. In model 2, parenchymal cells that arise from the fetal mesothelium in development contribute to repair. To what degree these models are involved in lung re- growth and remodeling in post-natal life will be further examined (Aim 3). We expect that completion of these studies will establish a firm foundation for future work in this new area of lung biology.

ABSTRACT We have identified a subset of ciliated cells (30%) in the mouse and human airways that express MIWI2, a protein only previously observed in the mouse testes where it functions to suppress transposon transcription. Newly generated ontogeny data along with global transcriptomic and gene ontology analyses of mouse MIWI2-positive (MPACs) and negative ciliated cells (non-MPACs) further support the concept of multiciliated cell heterogeneity and our overall hypothesis: MIWI2-positive expression distinguishes 2 distinct ciliated cell lineages in the airway. For the first time, these data hint at functionally distinct multi-ciliated cell subsets and suggest new lineage models for the airway epithelium. In view of the broader implications of this work for the field, our over-riding objective and focus in this revised grant are to develop and elucidate a basic understanding of the differential biology of these 2 subtypes of ciliated cells in the mouse airway. As a result, many of our studies are now deliberately and logically directed at characterizing the ontogeny, half-life, and origins of MPACs relative to non-MPACs. Our data also indicate that ciliated cells play a distinct role in regulating inflammation during injury, a function not generally ascribed to this cell type. Determining the relative role of MPACs and non-MPACs and ciliated MIWI2 in regulating inflammatory states in the lung are thus important sub-goals of this proposal. In our view, identifying immune-regulatory roles for ciliated cells has not been sufficiently addressed since most of the research on this cell type has traditionally concentrated on their mechanical contributions to mucus clearance. As such, this direction lends a deeper and more expansive significance to our proposed studies. By using several unique genetic mouse models that sustain the identification, tracking, and fate of ciliated cells, including MPACs, we propose to examine our hypothesis and meet our goals in 3 Aims. In Aim 1 we well establish the ontogeny of MPACs, their lineage relationship to non-MPACS and their half-life during lung homeostasis. In Aim 2, we will identify the origin of new MPACs that arise during airway remodeling after lung infection and during MPAC- specific cell regeneration. In Aim 3, we will determine the relative roles of MIWI2 protein and MPACs in the host response to early lung inflammation and infection. Together, these studies will forge new directions in the fundamental biology of the airway by establishing a new understanding of epithelial cell lineages and by integrating and connecting the fields of Piwi proteins, inflammation, and multiciliated cells of the lung.

ABSTRACT The overall objective of this T35 proposal is to provide meaningful ten week summer research experiences for twelve first year medical students in settings characterized by excellence in investigation and excellence in mentoring. Our long-term goal is to establish a foundation for future physician- scientist careers. To do this, we plan to focus this program on research relating to heart, lung, and blood disorders. This strategy takes advantage of the long history of funded and excellent research in these areas on our campus and affiliated hospitals. We will utilize our 5 NHLBI funded T32 faculty for research placements. These T32s are focused in the following areas: 1) lung biology 2) regenerative medicine 3) cardiovascular epidemiology 4) blood diseases, and 5) cardiovascular biology. This strategy ensures that T35 faculty are committed to mentorship and engaged in superior research, and provides a rich portfolio of topics and experiences for students to choose from. To enter this T35 program, interested students will submit a short research proposal in collaboration with a faculty mentor for review by a faculty/student committee. Once accepted, students will be expected to participate in the scholarly activities of their home T32 program, including seminars, presentations, lab meetings etc. This will be supplemented by weekly seminars to discuss RCR, critical thinking, and presentation skills. During the following winter, students will present their research projects at a campus-wide research symposium. Students will be encouraged to work with their mentors to prepare an abstract for submission to a national research meeting. Longitudinal scholarly experiences via continued work with their mentors for the remaining three years of medical school is an option for T35 participants and will fulfill a BUSM research track requirement. Students achieving this will earn a special designation upon graduation. In order to assess the effect of our T35 program, students will be given an ORCID so that future scholarly activities can be monitored. In addition, there are built in defined metrics to guide programmatic improvement and assess overall impact. In sum, we strongly believe that this new T35 program will provide meaningful research experiences that will propel careers as physician-scientists.