Fibronectin in immune responses in organ transplant recipients.

The immune response to an organ allograft involves perpetuation of T cell infiltration and activation. Advances in understanding the mechanisms of T cell activation have placed particular emphasis on the interactions between the T-cell receptor and antigen presenting cells, with little reference to the fact that in vivo activation occurs in the physical context of extracellular matrix proteins (ECM). Indeed, the possibility that ECM proteins may have a determining role in lymphocyte adhesion and tissue localization and function is now becoming more appreciated in view of growing evidence indicating that integrins and other T cell antigens bind ECM components, with some of these components exerting synergistic effects on T-cell activation. This review focuses on the importance of interactions between lymphocytes and fibronectin, a prominent ECM component, for cell migration and function in organ allograft recipients. It explores novel therapeutic approaches based on the assumption that fibronectin represents an active element in the process of T cell activation in the immune cascade triggered by organ transplantation

Cell-Based Multi-Parametric Model of Cleft Progression during Submandibular Salivary Gland Branching Morphogenesis

Cleft formation during submandibular salivary gland branching morphogenesis is the critical step initiating the growth and development of the complex adult organ. Previous experimental studies indicated requirements for several epithelial cellular processes, such as proliferation, migration, cell-cell adhesion, cell-extracellular matrix (matrix) adhesion, and cellular contraction in cleft formation; however, the relative contribution of each of these processes is not fully understood since it is not possible to experimentally manipulate each factor independently. We present here a comprehensive analysis of several cellular parameters regulating cleft progression during branching morphogenesis in the epithelial tissue of an early embryonic salivary gland at a local scale using an on lattice Monte-Carlo simulation model, the Glazier-Graner-Hogeweg model. We utilized measurements from time-lapse images of mouse submandibular gland organ explants to construct a temporally and spatially relevant cell-based 2D model. Our model simulates the effect of cellular proliferation, actomyosin contractility, cell-cell and cell-matrix adhesions on cleft progression, and it was used to test specific hypotheses regarding the function of these parameters in branching morphogenesis. We use innovative features capturing several aspects of cleft morphology and quantitatively analyze clefts formed during functional modification of the cellular parameters. Our simulations predict that a low epithelial mitosis rate and moderate level of actomyosin contractility in the cleft cells promote cleft progression. Raising or lowering levels of contractility and mitosis rate resulted in non-progressive clefts. We also show that lowered cell-cell adhesion in the cleft region and increased cleft cell-matrix adhesions are required for cleft progression. Using a classifier-based analysis, the relative importance of these four contributing cellular factors for effective cleft progression was determined as follows: cleft cell contractility, cleft region cell-cell adhesion strength, epithelial cell mitosis rate, and cell-matrix adhesion strength

αv integrins: key regulators of tissue fibrosis

Chronic tissue injury with fibrosis results in the disruption of tissue architecture, organ dysfunction and eventual organ failure. Therefore, the development of effective anti-fibrotic therapies is urgently required. During fibrogenesis, complex interplay occurs between cellular and extracellular matrix components of the wound healing response. Integrins, a family of transmembrane cell adhesion molecules, play a key role in mediating intercellular and cell-matrix interactions. Thus, integrins provide a major node of communication between the extracellular matrix, inflammatory cells, fibroblasts and parenchymal cells and, as such, are intimately involved in the initiation, maintenance and resolution of tissue fibrosis. Modulation of members of the αv integrin family has exhibited profound effects on fibrosis in multiple organs and disease states. In this review, we discuss the current knowledge of the mechanisms of αv-integrin-mediated regulation of fibrogenesis and show that the therapeutic targeting of specific αv integrins represents a promising avenue to treat patients with a broad range of fibrotic diseases

Isolation and characterization of a common nuclear matrix protein and other studies

The nuclear matrix, the RNA-protein skeleton of the nucleus, is known to play functional and structural roles within the cell. My thesis investigated the roles of the nuclear matrix proteins under various circumstances. First, the rat prostate cancer cell line (MLL) was injected into various sites within the rat. Tumors were isolated and nuclear matrix proteins isolated and analyzed by two-dimensional electrophoresis. Upon comparison, distinct nuclear matrix protein differences were observed in all of the organ sites that were investigated suggesting that the extracellular matrix, as well as growth factors and hormones, may be important in directing the production of nuclear matrix proteins. Second, nuclear matrix proteins from various cell lines representing the four classes of human lung carcinoma were analyzed. Upon comparison, it was observed that there were at least two classes of nuclear matrix proteins present. The majority of the nuclear matrix proteins were common within each lung cancer category, while other nuclear matrix proteins were found to be different. These nuclear matrix protein differences may be due to the various classes of phenotypic and genotypic distinct classes of lung cancer. Lastly, several reproducible nuclear matrix proteins isolated from the human breast MCF10A cell line were characterized. Although cDNA library screening proved unsuccessful, antibodies toward one of these proteins (KP3) provided further information into the function of this protein. Western and immunocytochemical analyses provided evidence to suggest that this protein was common and could be found in all cell lines and tissues that were analyzed. In addition, further analyses suggested that this protein could play an important role(s) in mitosis. Multidrug resistance (MDR) was also investigated in various human and rat prostate cancer cell lines. The majority of the cell lines analyzed did express an increased MDR phenotype when compared to the MDR negative controls. Two rat prostate cell lines (MLL and AT3) where selected and grown in increasing concentrations of doxorubicin to enhance the MDR phenomenon. MLL and AT3 MDR cell lines had an increased MDR phenotype when compared to their normal counterparts

Matrix metalloproteinases in liver injury, repair and fibrosis.

The liver is a large highly vascularized organ with a central function in metabolic homeostasis, detoxification, and immunity. Due to its roles, the liver is frequently exposed to various insults which can cause cell death and hepatic dysfunction. Alternatively, the liver has a remarkable ability to self-repair and regenerate after injury. Liver injury and regeneration have both been linked to complex extracellular matrix (ECM) related pathways. While normal degradation of ECM components is an important feature of tissue repair and remodeling, irregular ECM turnover contributes to a variety of liver diseases. Matrix metalloproteinases (MMPs) are the main enzymes implicated in ECM degradation. MMPs not only remodel the ECM, but also regulate immune responses. In this review, we highlight some of the MMP-attributed roles in acute and chronic liver injury and emphasize the need for further experimentation to better understand their functions during hepatic physiological conditions and disease progression

Tissue engineering: construction of a multicellular 3D scaffold for the delivery of layered cell sheets.

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.(2,3) Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.(4) As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.(2,5,6) A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the "tissue". Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct

The Use of Organ Cultures to Study Vessel Wall Pathobiology

Organ culture of the vessel wall is an useful in vitro method to study vascular cell biology. The intact vessel allows for the study of cell-cell and cell-substratum interactions including the structure and function of the vessel wall matrix. Long term organ cultures of porcine aorta show that neointimal formation is due primarily to cell proliferation of pre-existing intimal smooth muscle cells. Neointimal formation in these cultures is more pronounced in the presence of an endothelium that is turning over. In endothelial wound repair studies, the endothelium of the organ culture shows some important differences when compared to tissue culture studies in monolayer culture. Thus, vascular organ cultures can be successfully used to study vessel wall biology in health and disease

The transmembrane protein fibrocystin/polyductin regulates cell mechanics and cell motility

Polycystic kidney disease is a disorder that leads to fluid filled cysts that replace normal renal tubes. During the process of cellular development and in the progression of the diseases, fibrocystin can lead to impaired organ formation and even cause organ defects. Besides cellular polarity, mechanical properties play major roles in providing the optimal apical-basal or anterior–posterior symmetry within epithelial cells. A breakdown of the cell symmetry that is usually associated with mechanical property changes and it is known to be essential in many biological processes such as cell migration, polarity and pattern formation especially during development and diseases such as the autosomal recessive cystic kidney disease. Since the breakdown of the cell symmetry can be evoked by several proteins including fibrocystin, we hypothesized that cell mechanics are altered by fibrocystin. However, the effect of fibrocystin on cell migration and cellular mechanical properties is still unclear. In order to explore the function of fibrocystin on cell migration and mechanics, we analyzed fibrocystin knockdown epithelial cells in comparison to fibrocystin control cells. We found that invasiveness of fibrocystin knockdown cells into dense 3D matrices was increased and more efficient compared to control cells. Using optical cell stretching and atomic force microscopy, fibrocystin knockdown cells were more deformable and exhibited weaker cell–matrix as well as cell–cell adhesion forces, respectively. In summary, these findings show that fibrocystin knockdown cells displayed increased 3D matrix invasion through providing increased cellular deformability, decreased cell–matrix and reduced cell–cell adhesion force

Biophysical regulation of stem cell behavior within the niche.

Stem cells reside within most tissues throughout the lifetimes of mammalian organisms. To maintain their capacities for division and differentiation and thereby build, maintain, and regenerate organ structure and function, these cells require extensive and precise regulation, and a critical facet of this control is the local environment or niche surrounding the cell. It is well known that soluble biochemical signals play important roles within such niches, and a number of biophysical aspects of the microenvironment, including mechanical cues and spatiotemporally varying biochemical signals, have also been increasingly recognized to contribute to the repertoire of stimuli that regulate various stem cells in various tissues of both vertebrates and invertebrates. For example, biochemical factors immobilized to the extracellular matrix or the surface of neighboring cells can be spatially organized in their placement. Furthermore, the extracellular matrix provides mechanical support and regulatory information, such as its elastic modulus and interfacial topography, which modulate key aspects of stem cell behavior. Numerous examples of each of these modes of regulation indicate that biophysical aspects of the niche must be appreciated and studied in conjunction with its biochemical properties

Modeling Progressive Fibrosis with Pluripotent Stem Cells Identifies an Anti-fibrotic Small Molecule.

Progressive organ fibrosis accounts for one-third of all deaths worldwide, yet preclinical models that mimic the complex, progressive nature of the disease are lacking, and hence, there are no curative therapies. Progressive fibrosis across organs shares common cellular and molecular pathways involving chronic injury, inflammation, and aberrant repair resulting in deposition of extracellular matrix, organ remodeling, and ultimately organ failure. We describe the generation and characterization of an in vitro progressive fibrosis model that uses cell types derived from induced pluripotent stem cells. Our model produces endogenous activated transforming growth factor β (TGF-β) and contains activated fibroblastic aggregates that progressively increase in size and stiffness with activation of known fibrotic molecular and cellular changes. We used this model as a phenotypic drug discovery platform for modulators of fibrosis. We validated this platform by identifying a compound that promotes resolution of fibrosis in in vivo and ex vivo models of ocular and lung fibrosis