Epithelial to mesenchymal transition (EMT) is an essential process during embryogenesis for the development of organ systems, including the heart and its vasculature. The development of both coronary vessels and heart valves depends on EMT. In this dissertation, we first present data demonstrating that increased oligosaccharide hyaluronan (o-HA) levels after EMT induction within atrioventricular (AV) valves leads to a decrease in EMT due to the induction of VEGF expression. Regulated EMT inhibition prevents the formation of hyperplastic valves. Next, we show that the proepicardium, which provides the precursor cells required for epicardial and coronary vessel development, migrates to the developing heart via direct contact of multicellular proepicardial villi to the developing myocardium. This shifts the paradigm from a migration consisting of floating cysts to one of direct contact and differential adhesion forces to form the initial epicardium. A subset of epicardial cells undergoes EMT, migrates into the developing heart, and differentiates into cardiac fibroblast, vascular endothelial, and smooth muscle cells. In order to more effectively study epicardial EMT in vitro, we developed several new methods for the in vitro study of coronary vessel development. We developed an improved protocol for isolating embryonic myocyte cells, for use in co-cultures with epicardial cells. This co-culture system allows investigation into the effects of myocyte derived soluble factors upon epicardial EMT and mesenchymal cell differentiation. We also present a protocol for isolating epicardial clonal colonies from an epicardial cell line derived from the ImmortoMouse. These clones provided direct evidence that the epicardium is a
Tight junctions (TJ) regulate the paracellular movement of ions, macromolecules and immune cells across epithelia. Zonula Occludens (ZO)-1 is a multi-domain polypeptide required for the assembly of TJs. MDCK II cells lacking ZO-1, and its homolog ZO-2, have three distinct phenotypes: Reduced localization of occludin and some claudins to the TJ, increased epithelial permeability, and expansion of the apical actomyosin contractile array found at the apical junction complex (AJC). However, it is unclear exactly which ZO-1 binding domains are required to coordinate these activities. We addressed this question by examining the ability of ZO-1 domain-deletion transgenes to reverse the effects of ZO-depletion. We found that the SH3 domain and the U5 motif are required to recruit ZO-1 to the AJC and that localization is a prerequisite for normal TJ and cytoskeletal organization. The PDZ2 domain is not required for localization of ZO-1 to the AJC, but is necessary to establish the characteristic continuous circumferential band of ZO-1, occludin and claudin-2. PDZ2 is also required to establish normal permeability, but is not required for normal cytoskeletal organization. Finally, our results demonstrate that PDZ1 is critical for the normal organization of both the TJ and the AJC cytoskeleton. Our results establish that ZO-1 acts as a true scaffolding protein and that the coordinated activity of multiple domains is required for normal TJ structure and function.
Lineage mapping studies have shown that progenitors for coronary smooth muscle enter the heart following epithelial to mesenchymal transition (EMT) of epicardial cells. Primary explant culture experiments show that loss of epicardial cell‐cell adhesion is a prerequisite for transcriptional activation of coronary smooth muscle cell (CoSMC) differentiation marker genes. Thus a majority of epicardial cells are specified for a CoSMC fate, but do not express that fate while resident within the intact epicardium. Our studies suggest that epicardial cell‐cell contacts activate Notch‐dependent pathways that oppose receptor tyrosine kinase (PDGFBB, FGF2)‐mediated signals for EMT and thereby maintain the CoSMC progenitor phenotype in the intact epicardium. Later steps in coronary development produce three distinct layers of artery wall. The outer layer is known as the adventitia and is composed of epicardium‐derived cells. We observed that formation of adventitia is accompanied by local activation of sonic hedgehog (Shh) signaling that is restricted to the adventitial layer. Moreover, we found a population of stem cell antigen 1‐positive cells (AdvSca1) resident within this novel domain of Shh signaling with intrinsic potential to differentiate to pericytes and CoSMCs. The role of AdvSca1 progenitors in coronary artery maturation, remodeling and disease will be discussed. Support: HL19242, HL93594.
Chronic ingestion of arsenic is associated with increased incidence of respiratory and cardiovascular diseases. To investigate the role of arsenic in early events in vascular pathology, C57BL/6 mice ingested drinking water with or without 50 ppb sodium arsenite (AsIII) for four, five, or eight weeks. At five and eight weeks, RNA from the lungs of control and AsIII-exposed animals was processed for microarray. Sixty-five genes were significantly and differentially expressed. Differential expression of extracellular matrix (ECM) gene transcripts was particularly compelling, as 91% of genes in this category, including elastin and collagen, were significantly decreased. In additional experiments, real-time RT-PCR showed an AsIII-induced decrease in many of these ECM gene transcripts in the heart and NIH3T3 fibroblast cells. Histological stains for collagen and elastin show a distinct disruption in the ECM surrounding small arteries in the heart and lung of AsIII-exposed mice. Immunohistochemical detection of α-smooth muscle actin in blood vessel walls was decreased in the AsIII-exposed animals. These data reveal a functional link between AsIII exposure and disruption in the vascular ECM. These AsIII-induced early pathological events may predispose humans to respiratory and cardiovascular diseases linked to chronic low-dose AsIII exposure.
The actin cytoskeleton is a dynamic structure necessary for cell and tissue organization, including the maintenance of epithelial barriers. The epithelial barrier regulates the movement of ions, macromolecules, immune cells, and pathogens, and is thus essential for normal organ function. Disruption in the epithelial barrier has been shown to coincide with alterations of the actin cytoskeleton in several disease states. These disruptions primarily manifest as increased movement through the paracellular space, which is normally regulated by tight junctions (TJ). Despite extensive research demonstrating a direct link between the actin cytoskeleton and epithelial permeability, our understanding of the physiological mechanisms that link permeability and tight junction structure are still limited. In this review, we explore the role of the actin cytoskeleton at TJ and present several areas for future study.
Many heart problems are associated with defects and disease of the coronary vessels; however coronary vessel development is not well understood. Coronary vessels originate from epicardial cells which come from the proepicardium (PE). At E9.0 in mice, PE cells migrate to and spread over the heart to form the epicardium. A subset of epicardial cells undergoes an epithelial to mesenchymal transformation (EMT) and migrates into the myocardium. These epicardial derived cells (EDC) differentiate into epithelial and smooth muscle cells of the coronary vessels. Signals required for this process are speculated to originate from the myocardium and endocardium, but there is no direct evidence for this activity. We have developed a novel in vitro system to directly address the question of “where do the signals required for early coronary vessel development originate?” In this system we co‐culture primary epicardial monolayers with embryonic myocytes. We detect an increase in epicardial EMT during co‐culture conditions compared to naïve epicardial cultures. We have identified several growth factors, including VEGF and TGF‐β2, which are produced by embryonic myocytes in vitro . Thus, our system allows for the identification of mediators responsible for epicardial EMT and, ultimately, EDC differentiation into the coronary vasculature. Supported by HL077493 and T32 HL07249.
We examined the role of interferon-γ (IFN-γ) in expression of chemokine mRNA and proteins in the brain during chronic infection with Toxoplasma gondii using BALB/c and BALB/c-background IFN-γ knockout (IFN-γ(-/-)) mice. BALB/c mice are genetically resistant to development of toxoplasmic encephalitis and establish a latent, chronic infection in the brain through IFN-γ-mediated immune responses. Amounts of mRNA for CXCL9/MIG, CXCL10/IP-10, CXCL11/I-TAC, CCL2/MCP-1, CCL3/MIP-1α, and CCL5/RANTES significantly increased in the brains of wild-type mice after infection. CXCL9/MIG, CXCL10/IP-10, and CCL5/RANTES mRNA were most abundant among these chemokines. An increase in amounts of mRNA for CXCL10/IP-10, CCL2/MCP-1, CCL3/MIP-1α, and CCL5/RANTES was also observed in the brains of IFN-γ(-/-) mice after infection, although CXCL10/I-10 and CCL5/RANTES mRNA levels in infected IFN-γ(-/-) mice were significantly lower than those of infected wild-type animals. Amounts of mRNA for CXCL9/MIG and CXCL11/I-TAC remained at the basal levels in infected IFN-γ(-/-) mice. When amounts of the chemokine proteins were examined in the brain homogenates of uninfected and infected mice of both strains, large amounts of CXCL9/MIG, CXCL10/IP-10, and CCL5/RANTES were detected only in infected wild-type animals. These results indicate that CXCL9/MIG, CXCL10/IP-10, and CCL5/RANTES are the chemokines predominantly induced in the brains of genetically resistant BALB/c mice during chronic infection with T. gondii, and their expression is dependent on IFN-γ.
Abstract We examined the role of IFN-γ in expression of chemokine mRNA and proteins in the brain during chronic infection with Toxoplasma gondii using BALB/c and BALB/c-background IFN-γ knockout (IFN-γ-/-) mice. BALB/c mice are genetically resistant to development of toxoplasmic encephalitis and establish a latent, chronic infection in the brain through IFN-γ-mediated immune responses. Amounts of mRNA for CXCL9, CXCL10, CXCL11, CCL2, CCL3, and CCL5 significantly increased in the brains of wild-type mice after infection. CXCL9, CXCL10, and CCL5 mRNA were most abundant among these chemokines. An increase in amounts of mRNA for CXCL10, CCL2, CCL3, and CCL5 was also observed in the brains of IFN-γ-/- mice after infection, although CXCL10 and CCL5 mRNA levels in infected IFN-γ-/- mice were significantly lower than those of infected wild-type animals. Amounts of mRNA for CXCL9 and CXCL11 remained at the basal levels in infected IFN-γ-/- mice. When amounts of the chemokine proteins were examined in the brain homogenates of uninfected and infected mice of both strains, large amounts of CXCL9, CXCL10, and CCL5 were detected only in infected wild-type animals. These results indicate that CXCL9, CXCL10, and CCL5 are the chemokines predominantly induced in the brains of genetically resistant BALB/c mice during chronic infection with T. gondii, and their expression is dependent on IFN-γ.
