Gregg L. Semenza - US grants

DESCRIPTION: (Adapted from investigator's abstract) The long-term goal of this research project is to understand the molecular mechanisms underlying the regulation of human erythropoietin (EPO) gene expression during development. The EPO gene encodes the polypeptide hormone which controls red blood cell production, and the gene is expressed in the fetal liver, adult kidney, and adult liver during stress erythropoiesis. Previous studies of human EPO gene expression in transgenic mice have localized cis-acting DNA regulatory elements which control human EPO gene expression in liver, but not kidney. Larger DNA fragments with more extensive 5'- and 3'-flanking regions will be microinjected into fertilized mouse eggs in order to generate transgenic mice that express the human EPO gene in kidney. The flanking sequences responsible for regulated EPO gene expression in liver and kidney will then each be joined to simian virus 40 early region coding sequences to construct a chimeric gene in which the transforming protein T antigen (Tag) is expressed only in EPO-producing cells of liver and kidney, respectively. Transgenic mice carrying the EPO-Tag transgene will be analyzed and attempts to establish permanent tissue-culture lines of EPO-producing cells will be made. Nuclease-sensitivity studies of the EPO gene will be performed using EPO-producing vs. heterologous cell lines to identify the precise location of DNA sequences controlling EPO gene expression. The importance of these sequences will be confirmed by constructing EPO transgenes for microinjection in which these specific regions have been deleted, and analyzing the resulting pattern of transgene expression in vivo. The trans-acting transcriptional factors which bind to these DNA regulatory elements will be identified using nuclear extracts of EPO-producing cells in gel-shift and DNA footprinting assays. To complement studies of transgenic mice, two human families will be studied in which erythrocytosis is inherited as an autosomal dominant trait and shows co-segregation with restriction fragment length polymorphisms that do not rule out linkage of the phenotype to the EPO gene. EPO gene DNA from affected family members will be amplified by the polymerase chain reaction and analyzed by nucleotide sequence determination to identify potential disease-causing mutations.

The process of human development is controlled by programs of regulated gene expression within the embryo. Birth defects are a major cause of morbidity and mortality in the pediatric population and thus represent a significant public health problem. Malformation syndromes result from gastric or environmental factors that disrupt developmentally-regulated programs of gene expression. Transcription factors are mediators of the genetic programs that define embryogenesis. Homeobox genes encode transcription factors containing a 60 amino acid domain that is capable of binding to DNA and these genes play essential roles in development. Msx-1 nd Msx-2 are homeodomain proteins that are expressed in developing cardiac, limb, and craniofacial structures during mammalian embryogenesis, especially at sites of important epithelio-mesenchymal inductive tissue interactions. Craniosynostosis, Boston type is a malformation syndrome with autosomal dominant inheritance that affects limb and craniofacial development. A mutation in the human MSX2 gene has been demonstrated in patients with this disorder that results in a substitution of histidine for an evolutionarily-conserved prokine residue at position 7 of the homeodomain. The long-term goal of the proposed research is to determine the role that MSX-1 and MSX-2 homeodomain proteins play in normal craniofacial development and the mechanisms by which mutations in the genes encoding these proteins may result in human craniofacial malformation syndromes. The specific aims of this project are (i) to establish the pathogenesis of craniosynostosis, Boston type by molecular biologic and embryologic analyses of mutant and wild-type Msx-2 protein expression in vitro within tissue culture cells and in vivo murine development;l and (ii) to determine whether disruption of normal Msx-1 expression during murine development also results in a craniofacial malformation syndrome. These studies will further our understanding of the role of Msx proteins in craniofacial development, provide model systems for the study of craniofacial malformations, and establish a crucial link between the function of Msx proteins as putative transcriptional regulatory factors and their role in directing craniofacial development during human embryogenesis.

Among known teratogens, ethanol represents the greatest public health problem in the U.S., playing an etiological role in approximately 5% of all congenital cardiac, central nervous system, and craniofacial malformations. Despite its enormous significance, little is known regarding the molecular pathogenesis of ethanol embryopathy. Cranial neural-crest derived cells (CNCC) are a major constituent of all developing craniofacial structures. Exposure of chick or mouse embryos to ethanol results in excessive craniofacial development. Increased expression of MSX2 and the signaling molecule BMP4 is associated with developmentally-programmed death of CNCC in the hindbrain. Over-expression of MSX2 results in craniofacial malformations strikingly similar to those associated with ethanol exposure. The goal of this project is to determine whether MSX1, MSX2, and/or BMP4 are involved in the excessive cell death associated with ethanol-induced craniofacial malformations. The Specific Aims are as follows: (1) To determine whether craniofacial malformations in MSX2 transgenic mice result from excessive death of MSX2- over-expressing cells. (2) To determine whether MSX1 over-expression affects survival of CNCC and subsequent craniofacial development in mouse embryos. (3) To determine whether ethanol exposure alters Msx1, Msx2, and/or Bmp4 gene expression in mouse embryos. (4) To determine whether ethanol affects survival of CNCC and subsequent craniofacial development in zebrafish embryos and whether these effects are mediated by BMP4 and/or MSX proteins. (5) To determine whether loss-of-function mutations at the Msx1 or Msx2 locus susceptibility to ethanol- induced craniofacial malformations in mice. (6) To identify genes that are transcriptionally regulated by MSX2 in CNCC of mouse embryos that over-express MSX2 or have been exposed to ethanol in utero. In addition to increasing our understanding of disease pathogenesis and of normal development, the identification of genes involved in this process may ultimately provide a diagnostic method for identifying pregnancies at increased risk for ethanol embryopathy.

myocardial ischemia /hypoxia; gene induction /repression; gene expression; transcription factor; biological signal transduction; cytoprotection; cardiac myocytes; oxygen tension; genetically modified animals; laboratory rat; laboratory mouse;

Humans and other mammals have a constant and absolute requirement for O2 and tissue oxygenation is maintained within a narrow physiologic range. In many disease states, however, O2 homeostasis is disrupted Hypoxia is a major factor contributing to the pathophysiology of heart attack, stroke, pulmonary hypertension, and other important causes of morbidity. The broad, long objective of the proposed research is to increase our understanding of the role of the transcriptional regulator hypoxia-inducible factor 1 (HIF-1) in the maintenance of cellular and systematic 02 homeostasis. HIF-1 is a heterodimeric basic-helix-loop- helix PAS transcription factor consisting of HIF-1alpha and HIF-1beta subunits. HIF-1alpha expression and HIF-1 transcriptional activity increase exponentially as cellular O2 concentration is decreased. Several dozen target genes that are transactivated by HIF-1 have been identified including those encoding erythropoietin, glucose transporters, glycolytic enzymes, and vascular endothelial growth factor. The products of these genes either increase O2 delivery or allow metabolic adaptation to reduced O2 availability. HIF-1 is required for cardiac and vascular development and embryonic survival. In fetal and postnatal life, HIF-1 is required for a variety of physiological responses to chronic hypoxia. The specific aims of this proposal are: to determine the role of HIF-1 in the pathogenesis of cerebral ischemia and analyze the expression of HIF-1 in ischemic kidney and liver; to determine the involvement of HIV-1 in wound healing; and to elucidate the signal transduction pathways by which insulin-like growth factor-1 receptor and V-SRC activity induce expression of HIF-1. The knowledge gained from the proposed experiments will be relevant to the treatment of many clinical conditions in which hypoxia or ischemia plays an important pathophysiologic role.

DESCRIPTION (provided by applicant): Humans and other mammals have a constant and absolute requirement for oxygen and tissue oxygenation is maintained within a narrow range. In many disease states, however, oxygen homeostasis is disrupted and hypoxia/ischemia is an important factor contributing to the pathophysiology of ischemic cardiovascular disease, chronic lung disease, and other major causes of mortality. The broad, long-term goal of the proposed research is to increase our understanding of the role of the transcriptional regulator hypoxia-inducible factor 1 (HIF-1) in the maintenance of oxygen homeostasis. In particular, the studies that will be performed in years 10-14 of R01HL55338 will focus on the role of HIF-1 as a master regulator of tissue vascularization. The knowledge gained from the proposed experiments will be relevant to the treatment of disorders associated with altered vascularization, particularly ischemic cardiovascular disease. The specific aims are: 1. To determine the role of HIF-1 in the vascular response to tissue ischemia. We will test the hypothesis that HIF-1 activity within ischemic muscle promotes angiogenic growth factor gene expression and vascularization. 2. To investigate the role of HlF-1 in vascular progenitor cells. We will test the hypothesis that HIF-1 activity in endothelial progenitor cells (EPCs) promotes their differentiation, recruitment, and survival, and stimulates EPC-mediated vascular remodeling in ischemic tissue. To delineate transcriptional networks regulated by HIF-1 in human arterial endothelial and smooth muscle cells. We will test the hypothesis that HIF-1 controls the expression of distinct batteries of genes in a cell type- specific manner and that this regulation is mediated via either direct DMA binding or indirectly via the regulation of other transcription factors.

Burn injuries represent a major public health problem, requiring medical attention for more than one million Americans annually. Despite therapeutic advances, non-healing burn wounds and excessive scarring still result in significant long-term physical and psychosocial morbidity. In this P20 Exploratory Center Grant application, we propose to perform clinical and pre-clinical research to study the role of endothelial progenitor cells (EPCs) in promoting burn wound healing. EPCs have been shown to contribute to vascularization and tissue repair in animal models of ischemia. Mobilization of EPCs into the peripheral blood has been demonstrated in burn wound patients. The proposed research will utilize: clinical resources of the Johns Hopkins Regional Burn Center, where hundreds of adults are treated each year;a mouse model of burn wound healing established by participating clinician-scientists;and expertise concerning the cellular and molecular mechanisms of angiogenesis induced by hypoxia/ischemia. The overall goal of the proposed research is to test the hypothesis that the mobilization of bone marrow-derived EPCs and their recruitment to burn wounds is a major determinant of the extent and quality of healing and that strategies designed to promote EPC mobilization and recruitment will promote burn wound healing. The proposed research is thus inherently translational. At the molecular level of analysis, we will focus on the role of hypoxia-inducible factor 1 (HIF-1), a transcription factor that functions as a master regulator of ischemiainduced angiogenesis, although its role in burn wound healing has not been investigated to date. Our highrisk strategy will involve attempting to mobilize and recruit EPCs to burn wounds by increasing the expression of HIF-1 and/or levels of cytokines encoded by HIF-1-regulated genes. To form the nucleus of the Johns Hopkins Center for Innovative Wound Healing Research, we have assembled an interactive multidisciplinary team with expertise in molecular and cellular biology, medical genetics, animal models of wound healing, and clinical burn wound care and research. This team will work together to perform a single project encompassing the innovative studies described above, which will provide the scientific foundation for clinical trials to be proposed in a subsequent P50 application, which will be submitted during year 3 of P20 funding and will lead to novel treatments to promote wound healing and prevent excessive scarring.

Coronary artery stenosis, resulting in impaired cardiac perfusion, ischemia, and the risk of myocardial infarction,[unreadable] is a major cause of morbidity and mortality in the U. S. population. There is currently tremendous interest in[unreadable] understanding the endogenous responses to ischemia and infarction. These studies may lead to the[unreadable] development of novel therapeutic strategies that protect the heart by promoting adaptive responses or by[unreadable] preventing maladaptive responses. Ischemia is characterized by deprivation of oxygen (hypoxia), metabolic[unreadable] substrates, and cytokines/survival factors, as well as accumulation of toxic metabolites. Despite the complex[unreadable] pathophysiology of ischemia, hypoxia alone is a sufficient stimulus to induce a variety of adaptive responses[unreadable] that protect against ischemia-reperfusion injury. An important mediator of these responses is hypoxia-inducible[unreadable] factor 1 (HIF-1), a transcription factor that regulates the expression of hundreds of genes in response to[unreadable] changes in cellular oxygenation. Among the known HIF-1 target genes are those encoding erythropoietin[unreadable] (EPO) and vascular endothelial growth factor (VEGF), which promote oxygen delivery to tissues by stimulating[unreadable] the production of red blood cells and blood vessels, respectively. HIF-1 target genes also encode survival[unreadable] factors, such as insulin-like growth factor 2 as well as EPO and VEGF, which can block apoptotic signaling[unreadable] induced by ischemia. HIF-1 also controls the expression of glucose transporters and glycolytic enzymes, which[unreadable] are required for anaerobic ATP production. In this proposal, we will investigate the role of HIF-1 and of proteins[unreadable] encoded by HIF-1 target genes, such as EPO, in promoting protection against cardiac ischemia-reperfusion[unreadable] injury. Aim 1 will investigate the mechanisms by which EPO protects the heart from injury following ischemia[unreadable] and reperfusion. Aim 2 will investigate the role of HIF-1 in mediating adaptive responses to cardiac ischemia[unreadable] and reperfusion. Aim 3 will investigate the mechanisms and consequences of the recruitment of bone marrowderived[unreadable] stromal cells to the ischemic heart,

Many human cancers contain regions of hypoxia, resulting in cell death and establishing a selection for cancer cells. Cancer cells adapt to the hypoxic microenvironment is through the activity of the transcription factor hypoxia-inducible factor 1 (HIF-1). HIF-1 also plays a critical role in tumor vascularization by activation of endothelial progenitors and cells. Project 1 will investigate the functional interactions between HIF-1 and extracellular matrix (ECM), both in cancer cells and in endothelial progenitors and cells. This is a unique perspective that takes into account how one aspect of the tumor microenvironment (02 concentration) regulates and interacts with another (mechanical properties of the ECM) to alter the cancer cell phenotype. The combined utilization of biophysical, biochemical, biological, computational, and engineering approaches that we propose will provide new insights into the mechanisms underlying metastasis. The Specific Aims are: (1) To determine whether alterations in mechanical properties of the ECM alter the phenotype of cancer cells.(2) To determine whether hypoxia and/or increased HIF-1 activity induces changes in mechanical properties of the ECM, which in turn alter the phenotype of cancer cells. (3) To determine whether alterations in mechanical properties of the ECM regulate HIF-1 activity, leading to alterations in cancer cell phenotype. (4) To determine whether changes in ECM mechanical properties alter the phenotype of endothelial progenitors and cells. (5) To determine whether HIF-1-induced changes in ECM mechanical properties alter the phenotype of endothelial progenitors and cells. Linkage to PS-OC: The research aims of Project 1 fit the overarching theme of the Center of the cooperative role of HIF-1 and EMC in the metastatic cascade; Aims 1 and 2 are synergistically connected to Aims 1-4 in Project 2 and Aims 1 and 2 in Project 3 for further research integration of the Center; all Students and Fellows in the Project will be enrolled in the Center Training Program; this project will make use of the resources provided by the Imaging Core, as well as the Administrative Unit of the Center; cell lines and micromechanical methods will be the same as those used in all projects; computational efforts will be shared among all projects.

DESCRIPTION (provided by applicant): The existence of hematopoietic stem-progenitor cells, which are present in the adult marrow and capable of self-renewal and differentiation into the progenitors that give rise to all types of mature blood cells, has been known for over half a century. More recently, endothelial stem-progenitor cells have also been identified. There remain several major obstacles to a more complete understanding of the molecular mechanisms underlying the self-renewal and differentiation of these stem-progenitor cells. First, these cells are present in low abundance and are difficult to purify from their more differentiated progeny. Techniques are needed to expand these cells, while maintaining their capacity for subsequent differentiation, in order that they might be better studied and utilized therapeutically. Second, in the case of endothelial stem-progenitors, thus far it has not been possible to establish a hierarchical lineage similar to what has been described for hematopoietic cells. Third, it is not clear whether the hematopoietic and endothelial stem-progenitors in adult marrow are themselves the descendants of a common stem cell, the hemangioblast. The molecular basis for development is the elaboration of discrete programs of gene expression in each cell type. Two central mechanisms for regulating gene expression are transcription factors, which determine the rate at which DNA sequences are transcribed into mRNA, and microRNAs, which determine the rate at which mRNAs are transcribed into protein. We hypothesize that there are key transcription factors and microRNAs which play critical roles in regulating the self-renewal and differentiation of hematopoietic and endothelial stem-progenitor cells. These cells are essential for hemangiogenesis, the formation of blood and blood vessels, respectively, which in turn are essential for the continuous delivery of O2 to all cells of the body. Within the marrow, undifferentiated cells are located along the endosteum, whereas more differentiated cells are located in proximity to the highly vascularized marrow cavity, from whence they enter the peripheral blood. The endosteal niche is hypoxic and may promote stem cell maintenance, whereas increased O2 levels in the vascular niche may promote proliferation and differentiation. We hypothesize that O2 functions as a developmental regulator for hemangiogenic cells, such that stem vs progenitor cell populations may require different O2 concentrations. The transcriptional regulator hypoxia-inducible factor 1 (HIF-1) mediates adaptive developmental and physiological responses to hypoxia and plays an essential, but only partially defined role, in hemangiogenesis. In this grant application, we propose to delineate the molecular mechanisms whereby HIF-1 and microRNAs regulate hemangiogenesis. By doing so, we hope to: better identify and characterize hemangiogenic progenitor cell lineages;direct the differentiation of stem and progenitor cells to desired cell fates;and develop new strategies for therapeutic utilization of these cells for in vivo hematopoiesis and vascularization.