Wnt1/ßcatenin injury response activates the epicardium and cardiac fibroblasts to promote cardiac repair.

Wnts are required for cardiogenesis but the role of specific Wnts in cardiac repair remains unknown. In this report, we show that a dynamic Wnt1/ßcatenin injury response activates the epicardium and cardiac fibroblasts to promote cardiac repair. Acute ischaemic cardiac injury upregulates Wnt1 that is initially expressed in the epicardium and subsequently by cardiac fibroblasts in the region of injury. Following cardiac injury, the epicardium is activated organ-wide in a Wnt-dependent manner, expands, undergoes epithelial-mesenchymal transition (EMT) to generate cardiac fibroblasts, which localize in the subepicardial space. The injured regions in the heart are Wnt responsive as well and Wnt1 induces cardiac fibroblasts to proliferate and express pro-fibrotic genes. Disruption of downstream Wnt signalling in epicardial cells decreases epicardial expansion, EMT and leads to impaired cardiac function and ventricular dilatation after cardiac injury. Furthermore, disruption of Wnt/ßcatenin signalling in cardiac fibroblasts impairs wound healing and decreases cardiac performance as well. These findings reveal that a pro-fibrotic Wnt1/ßcatenin injury response is critically required for preserving cardiac function after acute ischaemic cardiac injury.

Epithelial barrier assembly requires coordinated activity of multiple domains of the tight junction protein ZO-1.

Tight junctions (TJs) 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 TJs, 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 crucial 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.

Depolymerized hyaluronan induces vascular endothelial growth factor, a negative regulator of developmental epithelial-to-mesenchymal transformation.

Cardiac malformations constitute the most common birth defects, of which heart septal and valve defects are the most frequent forms diagnosed in infancy. These cardiac structures arise from the endocardial cushions through dynamic interactions between cells and the extracellular matrix (cardiac jelly). Targeted deletion of the hyaluronan synthase-2 (Has2) gene in mice results in an absence of hyaluronan (HA), cardiac jelly, and endocardial cushions, a loss of vascular integrity, and death at embryonic day 9.5. Despite the requirements for Has2 and its product, HA, in the developing heart, little is known about the normal processing and removal of HA during development. Cell culture studies show that HA obtains new bioactivity after depolymerization into small oligosaccharides. We previously showed reduction in Has2 expression and diminished presence of HA at later stages of heart development as tissue remodeling formed the leaflets of the cardiac valves. Here we show that small oligosaccharide forms of HA (o-HA) act antagonistically to developmental epithelial-to-mesenchymal transformation (EMT), which is required to generate the progenitor cells that populate the endocardial cushions. We further show that o-HA induces vascular endothelial growth factor (VEGF), which acts as a negative regulator of EMT. This is the first report illustrating a functional link between oligosaccharide HA and VEGF. Collectively, our data indicate that following endocardial cell EMT, native HA is likely processed to o-HA, which stimulates VEGF activity to attenuate cardiac developmental EMT.

Arsenic-induced decreases in the vascular matrix.

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 alpha-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.

Regulation of epithelial permeability by the actin cytoskeleton.

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.

Predominant interferon-γ-mediated expression of CXCL9, CXCL10, and CCL5 proteins in the brain during chronic infection with Toxoplasma gondii in BALB/c mice resistant to development of toxoplasmic encephalitis.

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.

An improved protocol for the isolation and cultivation of embryonic mouse myocytes.

In vitro cultures of cardiomyocytes have proven to be a useful tool for toxicological, pharmacological, and developmental studies, as well as for the study of the cellular and molecular mechanisms responsible for proper myocyte function. One deficient area of research is that of myocyte proliferation. Cardiomyocyte proliferation dramatically diminishes soon after birth and has a very limited occurrence within the adult heart, thus limiting the use of adult cells for proliferation studies. An improved understanding of the requirements for myocyte proliferation will allow for the development of better approaches to repair damaged heart tissue. Here, we provide a protocol for the reliable isolation of embryonic mouse myocytes. These myocytes behave similarly to those in vivo, including their ability to proliferate, providing an ideal system for the study of cardiomyocyte proliferation.

Differential growth and multicellular villi direct proepicardial translocation to the developing mouse heart.

In the mammalian system the proepicardium (PE) arises from mesothelium of the septum transversum before translocation to the heart where it forms the epicardium and progenitor cells of the coronary vessels. Despite its importance, the process in which PE cells translocate to the myocardium in mammals is not well defined. The current paradigm states that cellular cysts of PE float across the pericardial space and contact the outer surface of the myocardium. This mechanism does not provide a satisfactory explanation for the directionality or localization of PE migration. To better define PE migration, we performed a detailed study of mouse PE development. We provide thorough documentation that redefines the size of the PE migratory field and the mechanism of migration. Our new model incorporates differential growth and direct contact between multicellular PE villi and the myocardium as mechanisms in formation of the epicardium.

Strains of Toxoplasma gondii used for tachyzoite antigens to stimulate spleen cells of infected mice in vitro affect cytokine responses of the cells in the culture.

Cytokine responses of lymphocytes against Toxoplasma gondii are usually studied by stimulating the cells from infected animals with tachyzoite lysate antigens (TLA) in vitro. We examined whether strains (genotypes) of the parasite used for TLA affect the production of IL-2, IL-4, IL-10 and IFN-gamma in the culture of spleen cells obtained from mice infected with the ME49 (type II) strain. The amounts of IL-2 and IL-4 detected in the culture supernatants following stimulation with TLA of the RH (type I) strain were significantly greater than those detected following stimulation with TLA of the ME49 strain at 2 and 4 weeks after infection, respectively. These results suggest that antigen variations among the strains of T. gondii affect cytokine production of lymphocytes of infected mice. Therefore, to obtain accurate information on cytokine production by immune lymphocytes of infected hosts, it is important to use the identical strain of T. gondii for both infection of animals and preparation of TLA to stimulate the lymphocytes in vitro.