James Varani - US grants

Laminin, a high molecular weight glycoprotein found in basement membranes, has been shown to mediate the attachment of several types of cells to basement membrane (type IV) collagen. Our preliminary studies have shown a direct correlation between the expression of an endogenous laminin-like substance on the surface of murine tumor cells and metastatic potential. It is hypothesized that the expression of this substance contributes to metastasis, possibly by facilitating attachment of the metastasizing cells to basement membranes. The overall goal of this work is to provide direct evidence to show that the expression of cell surface laminin contributes to metastatic potential. Two approaches will be used to do this. In the first approach we will examine uncloned heterogeneous murine tumors for differences in expression of cell surface laminin, isolating and establishing laminin-positive and laminin-deficient cell lines from these populations. Three methods will be used to do this. These will include: (1)\complement-mediated cytotoxicity in the presence of affinity-purified antilaminin antibodies; (2)\flow cytometry; and (3)\collagen attachment. The laminin-positive and laminin-deficient cell lines isolated by these means will then be compared with regard to tumorigenicity and metastasis formation. In a second approach we will add exogenous laminin to the laminin-deficient cells. The laminin-treated cells will then be compared with untreated control cells with regard to in vivo behavior. The significance of this research lies in the fact that it will provide direct evidence to determine whether or not the expression or cell surface laminin by tumor cells contributes to their potential for malignancy. (A)

The overall goal of the proposed studies is to elucidate cellular and molecular regulatory mechanisms that underlie acquisition of the invasive phenotype in a human skin organ culture model of invasion. In this model, exposure of organ-cultured skin to certain exogenous growth factors (notably epidermal growth factor or hepatocyte growth factor) results in erosion of the dermal-epidermal basement membrane and invasion of the underlying stroma by the epithelium. Acquisition of the invasive phenotype is accompanied by elaboration of increased amounts of matrix metalloproteinase-9 (MMP-9) (92-kD gelatinase B/type IV collagenase) and induction of motility in the epithelial cell population, and by up-regulation of collagenolytic activity in the adjacent stroma. It is hypothesized that exposure of the target tissue to the exogenous growth factors initiates signal-transduction through two mitogen-activated protein kinase (MAPK) cascades in the epidermis, and that the activation of these pathways results in MMP-9 elaboration and in the induction of keratinocyte motility. It is hypothesized further that stimulated epithelial cells elaborate factors that induce production of MMP-1 (interstitial collagenase) by adjacent fibroblasts. Together, these events bring about invasion. In Specific Aim I of the proposed studies, the goal will be to define the intracellular signaling events that are required for up-regulation of MMP-9 production and for stimulation of cell motility in the epidermis. A combination of analytical experiments and interventional approaches will be used to define the relevant signaling events and to interfere with their expression. In Specific Aim II, studies will be carried out to demonstrate that invasion-associated MMP-1 elaboration by dermal fibroblasts is a result of interaction of dermal fibroblasts with products of growth factor-stimulated epithelial cells (rather than with the exogenous growth factors themselves); and to assess intracellular signaling events that are required for fibroblast up-regulation of MMP-1.

DESCRIPTION (adapted from the application) Diabetes remains one of the leading causes of lower limb amputation and is a contributing factor in up to 70 percent of the greater than 55,000 amputations annually. In the majority of diabetic subjects, the underlying factor which contributes to eventual amputation is the diabetic foot ulcer. The overall goal of this application is to determine the capacity of topical retinoid treatment to induce changes in the skin of diabetic patients that would reduce the incidence of ulcer formation and improve healing of wounds when they did occur. We know from recent studies that topical retinoid treatment improves histological structure and biochemical function of skin damaged by age and/or excessive exposure to solar radiation. Since diabetic skin demonstrates the same atrophic changes seen in aged human skin, it might be possible to improve structure and function in diabetic skin with the same approach. If one can improve the structure and function of diabetic skin in a similar fashion, such skin would be (in theory) more resistant to ulcer formation, and would heal better if wounding did occur. A three-part approach is proposed to achieve the overall goal of this application. We will in Specific Aim I assess fibroblast growth, connective tissue synthesis, matrix metalloproteinase (MMP) elaboration, vascular development and reactivity and indices of oxidative stress in hip skin from diabetic patients, including those with microangiopathic complications, and evaluate the effects of topical retinol treatment on these parameters. In Specific Aim 2, the goal will be to assess fibroblast growth, connective tissue synthesis, MMP elaboration, vascular development and reactivity and indices of oxidative stress in diabetic skin predisposed to the development of ulceration utilizing organ culture techniques. The effects of retinol treatment on these parameters will be assessed. Specific Aim 3 will make use of a rodent model of diabetes. Here we will determine the effects of topical retinol treatment on dermal structure/function in streptozotocin diabetic (STZ-D) rats and compare abrasion wound formation and healing in control rats, STZ-D rats and STZ-D rats that have been pretreated with topical retinol.

DESCRIPTION (provided by applicant): Studies conducted with matrix metalloproteinase-3 (MMP-3; stromelysin-1) gene-deleted (MMP-3- /-) animals have indicated an important pro-inflammatory role for this enzyme in acute lung injury, but the mechanism by which stromelysin-1 contributes to the disease process is not well understood. MMP-3 may play a direct role in damage to the alveolar wall. Damage to the alveolar wall, in and of itself, may be sufficient to facilitate lung injury. Alternatively, MMP-3 may promote lung injury by contributing to the generation of factors that stimulate neutrophil recruitment to the lung. Neutrophil chemotactic factors derived from non-collagenous components of the extracellular matrix as well as chemotactic cytokines elaborated by macrophages may be differentially elaborated in MMP-3 -/- animals as compared to normal controls. The overall goal of the proposed research is to evaluate the possible mechanisms in order to understand, specifically, how MMP-3 contributes to acute lung injury. In Specific Aim I, we will delineate the cellular sources of MMP-3 in the lungs of normal mice and determine how MMP-3 levels change during acute lung injury. In normal and MMP-3 -/- mice we will assess the production of several other MMPs that are known to play a role in inflammation and will concomitantly evaluate the production of MMP inhibitors under the same conditions. It is important to determine if there are compensatory changes in other MMPs or MMP inhibitors in animals lacking MMP-3. In specific Aims II and III we will utilize an in vitro model of an alveolar wall to directly assess the role of MMP-3 in damage to the alveolar wall and the role of MMP-3 in neutrophil migration across the alveolar wall. Studies will be conducted under conditions that lead to acute lung injury in intact animals and under conditions that result in neutrophil migration across the alveolar wall occurs but that do not lead to tissue damage, per se. Finally, in Specific Aim IV, we will assess the role of MMP-3 in alveolar wall damage in vivo under conditions that lead to acute lung inflammation or that lead to neutrophil influx into the alveolar space without tissue damage. These studies will provide an overall understanding of the mechanism(s) by which MMP-3 contributes to acute lung injury.

DESCRIPTION (provided by applicant): The studies proposed here will examine a red marine algae extract for ability to inhibit the outgrowth of abnormal colonic epithelial cells. The source of the extract is the North Atlantic red algae, Lithothamnium coralliodides. The extract is rich in Ca2+ but also contains detectable levels of approximately 70 other minerals. Two pivitol questions will be addressed in this study. First, does the marine algae extract demonstrate efficacy against colonic hyperplasia, colonic polyps and colon carcinoma when used in a 15-month dietary study with genetcally "normal" mice on a high-fat Western stye diet, and is the extract more effective than inorganic Ca2+ alone? Second, is the extracellular calcium-sensing receptor (CaSR) the critical target for the chemopreventive effects of the algae extract? To address the first question, wild-type (C57bl/6) mice will be maintained for 15 months on a high fat diet that is known to result in the development of multiple colonic polyps and, in some mice, colon cancer (Nemark diet). Some mice on the high fat diet will also receive the algae extract. As controls, other mice will be fed the high fat diet supplemented with inorganic Ca2+ (calcium carbonate). The hypothesis on which the proposed studies are based is that the algae extract will provide more effective colon chemoprevention than inorganic Ca2+ alone. To address the second question, we will assess expression of CaSR in a series of human colon cancer cell lines maintained in the presence or absence of the algae extract. Some of the lines are known to be defective in their response to extracellular Ca2+ alone. We will compare growth arrest and differentiation of the Ca2+ - responsive and non-responsive cells to the marine algae extract. In additional experiments, we will use an siRNA approach to down-regulate CaSR expression in the colon cancer cell lines and determine if CaSR expression is critical for growth inhibition mediated by the algae extract. The results of these experiments will provide insight into cellular and molecular basis of colon cancer chemoprevention by the algae extract and will provide direction for subsequent clinical studies (if warranted). PUBLIC HEALTH RELEVANCE: The overall goals of the studies proposed in this application are to determine if a red marine algae extract that contains detectable levels of up to 70 different minerals has the capacity to suppress abnormal colon epithelial cell growth in mice on a high-fat, Western-style diet, do determine if the high-mineral - containing algae extract is more effective than calcium alone and to determine if the calcium-sensing receptor is the critical target for the growth modulating activities of the algae extract.

DESCRIPTION (provided by applicant): Abstract the long-term goal of the research proposed here is to develop methods for the isolation and in vitro maintenance of intact colonic crypts from histologically normal human colon tissue and from adenomatous polyps / malignant tumors. The isolated colonic crypts maintained in culture will provide a unique culture system for examining the effects of potential chemopreventive agents in a preclinical setting as well as for examining various aspects of growth regulation in the colon. The same culture system will, hopefully, prove useful for isolation and characterization of stem cells from normal and aberrant crypts. To achieve the goals of this proposal, we will do the following: Specific Aim: Isolate crypts from histologically normal human colon tissue and from adenomatous polyps; develop culture conditions that maintain crypt histological structure and biochemical function and that allow for progression of the histologically-normal crypts through proliferation, differentiation an apoptosis in a spatially and temporally appropriate manner, while allowing for continued growth and expansion of the tissue derived from premalignant adenomatous polyps.

ABSTRACT Epidemiological and interventional studies have demonstrated an inverse relationship between calcium intake and colon polyp formation. Recent meta-analysis of the calcium chemoprevention literature indicates that the protective effects of calcium extend to colorectal cancer, itself. However, the effects are modest. It has been estimated that a reduction in tumor formation of approximately 8% might be achieved for each 300 mg per day increase in calcium intake (over a baseline of 1000 mg per day). The proposed studies will test the hypothesis that a combination of calcium and a mix of cationic trace elements will be more effective than calcium alone at suppressing the growth of human colonic adenomas (colon polyps). For the proposed studies, we will utilize human colon polyp tissue maintained in enteroid culture. We have developed a culture system to grow the premalignant human colon tissue using a minimally supplemented culture medium containing only epidermal growth factor and pituitary extract as growth promoters. Adenoma tissue in enteroid culture from 21 different cultures (representing 17 subjects) is now ?banked away? as frozen stocks. DNA sequencing and bioinformatic data identifying the presence of mutations thought to influence colon polyp growth are available for each established culture. Phenotypic information (immuno-histochemical marker data) is also available for each of the premalignant tumors. In the proposed studies, enteroid cultures will be maintained under control conditions or exposed to increasing concentrations of calcium ? either alone or in conjunction with additional trace elements. The effects of each intervention will be evaluated over time by assessing phenotypic markers of growth and differentiation. RNA sequencing and proteomic display differences will be used to identify global changes induced by each intervention. The use of colon enteroid cultures provides a way to determine directly if human colon polyp growth can be more effectively modulated by calcium in conjunction with additional trace elements than by calcium alone. By using cultures established from tumors of several different subjects, we are in position to determine the variability in responsiveness to each intervention as a first step toward understanding the basis for variation in response among individuals.