Inflammation is an organism's response to infection or injury. Although inflammation is largely a reparative response, severe acute inflammation or persistent inflammation can also lead to tissue damage and organ dysfunction. In fact, inflammation is now recognized to be a critical pathologic component underlying a wide variety of diseases ranging from acute septic shock to chronic inflammatory diseases such as atherosclerosis, asthma, and rheumatoid arthritis, and cancer. Furthermore, excessive fibrosis - part of the healing response after inflammation, which entails the laying down of "scar-like" tissue in place of injured tissue - can impede tissue function.
Aberrant function of pathways involved in the development of the bone-marrow derived inflammatory cells can lead to the development of myeloproliferative disease, myodysplastic disease and leukemia. The overall goal of the Puré laboratory is to gain insights into the development of inflammatory cells and the molecular and cellular basis of inflammation and fibrosis, and to understand how the mechanisms involved in these processes contribute to leukemogenesis, and to the microenvironment of tumors and thereby impact tumor cell growth and metastasis.
Highlights of Recent Research Advances
CD44 in inflammation and fibrosis.
CD44 is a widely expressed cell surface adhesion molecule that mediates cell-cell and cell-matrix interactions. The turnover of a major glycosaminoglycan component of extracellular matrices, hyaluronan, which is a ligand of CD44, is also dependent on CD44, particularly CD44 expressed on mesenchymal/stromal cells. Antibody blocking studies and studies of CD44-deficient mice conducted by the Puré lab demonstrated that CD44 contributes to the recruitment of inflammatory cells to sites of inflammation and stimulates the production of inflammatory mediators by infiltrating leukocytes. They demonstrated that these functions of CD44 contribute, for example, to the two most devastating forms of cardiovascular disease in mouse models of atherosclerosis and stroke. Yet a comparison of the inflammation and fibrosis induced in response to certain forms of injury in wild-type and CD44 mice, suggests that CD44 can also be required for the resolution of inflammation and progression to a healing fibrotic response.
Recent studies from the Puré lab suggest that this apparant paradox may be explained by the role of CD44 in promoting inflammatory cell recruitment and activation versus its role in regulating gene expression, cell growth and cellular differentiation of tissue mesenchymal cells such as fibroblasts and smooth muscle cells, and the role of CD44 in matrix deposition and turnover, all of which contribute to the fibrotic response. To further dissect the role of CD44 in inflammation and fibrosis, the Puré lab is generating tissue specific knock-outs of CD44. Current goals of the Puré lab include determining the impact of deletion of CD44 from specific inflammatory cells, endothelial cells, fibroblasts, and smooth muscle cells, in mouse models of atherosclerosis and irradiation-induced lung fibrosis, and to determine the mechanisms by which CD44 regulates these important disease associated processes.
Role of tumor cell and stromal cell CD44 in cancer.
CD44 has been implicated in tumor growth and metastasis in animal models and in human. However, the role of CD44 in tumor progression appears to be complex and the roles of tumor cell and host cell CD44 have not been distinguished. Furthermore, the mechanisms by which CD44 regulates tumor progression are not known. The Puré lab has investigated the structure and function of CD44 on epithelial-derived tumor cells and demonstrated that proteolytic processing of CD44 leads to the incorporation of the liberated extracellular domain of CD44 as an integral component of the extracellular matrix. Furthermore, both the transmembrane and soluble forms of CD44 can modulate tumor growth and migration. These results, mostly based on in vitro studies, are currently being extended in the Puré lab where they are studying the impact of tumor cell CD44 on tumor growth and metastasis.
In addition to intrinsic mechanisms that promote tumor cell development and tumor progression, it is now appreciated that the tumor microenvironment, composed of infiltrating leukocytes, stromal fibroblasts and extracellular matrix, is also a critical determinant of tumor progression. Based on the fact that CD44 plays an important role in matrix turnover and recent evidence obtained in the Puré lab that CD44 plays an important role in regulating the growth and differentiation of stromal fibroblasts, they are also investigating the role of CD44 on tumor associated fibroblasts on tumor growth and progression.
Identification of FAPα as a potentially functionally important marker and therapeutic target in fibrosis and epithelial-derived tumors.
FAPα is a cell surface serine protease of the dipeptidyl peptidase IV family that is specifically induced on reactive fibroblasts in epithlelial-derived tumors. Recent studies from the Puré lab and others indicate that FAPα is also a potential diagnostic marker for irreversible fibrosis in for example, lung and liver. Its restricted expression pattern, and the potential of its enzymatic activity to modify extracellular matrix and thereby the microenvironment, has generated interest in the potential of FAPα as a therapeutic target in fibrosis and cancer. Recent results from the Puré lab indicate that tumor initiation/growth is inhibited in FAPα-deficient mice and in wild-type mice treated with a FAPα inhibitor. Studies are being conducted to determine the mechanisms by which FAPα promotes tumor progression.
In addition, genetic engineering technology is being used to express fluorescent/ luminescent probes, a conditional suicide gene, cre recombinase, and an enzymatically dead mutant of FAPα, under the control of the endogenous FAPα promoter. These genetically engineered mice will provide the tools necessary to a) non-invasively image stromal cells during the course of tumor progression using bioluminescence, and by 2-P microscopy, image the interactions between reactive stromal cells and tumor cells for the first time; b) conditionally ablate reactive stromal fibroblasts to determine their requirement to support different stages of tumor progression; c) manipulate gene expression specifically in tumor stromal cells; and d) determine the impact of FAPα enzymatic activity on the extracellular matrix and tumor progression.
Although the requirement for reactive stromal fibroblasts to support tumor survival/growth and progression and their role in fibrotic diseases such as idiopathic or irradiation-induced lung fibrosis and liver cirrhosis is appreciated, the differentiative program involved in the evolution of this cell type within tumors and injured tissue is not well understood. FAPα is arguably the best markers for fibroblasts that have undergone the differentiative program giving rise to these functionally unique reactive stromal cells. Characterization of the cis- and transacting-factors that determine the tissue specific and inducible expression of FAPα will therefore be studied to gain insight into the molecular basis underlying this differentiative program. Interestingly, deficiency in CD44 appears to prevent the induction of FAPα. The mechanism by which CD44 regulates fibroblast differentiation is therefore also be investigated by the Puré lab as described above.
Role of fatty acid metabolism, lipid mediators, and reactive oxygen and reactive nitrogen species in hematopoietic cell development, leukemogenesis and inflammation.
Over the past decade, the Puré lab has contributed to our understanding of how a number of inflammatory pathways contribute to atherosclerosis. They were the first to demonstrate that in contrast to the atheroprotective role of endothelial nitric oxide synthase, that inducible nitric oxide synthase (iNOS) promotes atherosclerosis. In addition, they established that CD44 promotes atherosclerosis by mediating leukocyte recruitment and activation as described above. In collaboration with the laboratory of Daniel Rader (University of Pennsylvania) they have demonstrated that an atheroprotective apolipoprotein, apoE, regulates inflammation. And in a series of studies conducted in collaboration with Dr. Garret FitzGerald (University of Pennsylvania) they have investigated the impact of fatty acid metabolism by cycloxygenase 2 (COX-2) and microsomal prostaglandin E synthase on atherogenesis and the development of aortic aneurysms. As part of these studies they have described the impact of these proatherogenic pathways on the composition of atherosclerotic lesions. Their results suggest one mechanism that may underly the increased risk of acute cardiovascular events associated with prolonged treatment of patients with COX-2 inhibitors and suggest that specific downstream effectors of COX may represent more appropriate targets for future generations of anti-inflammatory drugs that may not carry the same risks as that associated with COX-2 inhibitors.
Unsaturated fatty acids (arachidonic acid and linoleic acid) are also subject to oxidative modification by lipoxygenases including, 12/15-LOX. Deficiency in 12/15-LOX protects mice against atherosclerosis which was previously attributed to its role in the oxidation of low density lipoproteins (LDL- or the bad cholesterol) and decreased production of inflammatory eicosanoids/prostanoids. The Puré lab demonstrated that 12/15-LOX also has the potential to contribute to atherosclerosis by mediating the production of type 1 inflammatory cytokines in atherosclerotic lesions. Specifically, they found that 12/15-LOX plays a unique role in selectively regulating the expression of members of the IL-12 family of cytokines (IL-12 and IL-23) that drive type I inflammatory responses.
They went on to demonstrate that 12/15-LOX mediates the expression of the p40 component of Il-12 and IL-23 in a cell type and stimulus dependent manner. Importantly, the 12/15-LOX-dependent pathway they discovered is unique with respect to its selectivity in regulating a subset of inflammatory genes and in being selectively invoked in macrophages.
Thus, they plan to extend these studies to test the hypothesis that this novel 12/15-LOX-dependent pathway preferentially contributes to chronic inflammation and macrophage dominated inflammatory responses and as such, may provide molecular targets for novel therapies to treat chronic inflammatory diseases without compromising the acute immune response to infection or injury. To date they have demonstrated that the deletion or inhibition of 12/15-LOX results in hyperactivity of a critical signal transduction pathway mediated by PI3-kinase and a defect in PI3-kinase-dependent regulation of the hematpoietic cell specific transcription factor IRF-8/ICSBP which regulates IL-12p40 expression. Our goals for the near future are to determine the mechanism by which 12/15-LOX regulates IRF-8/ICSBP function and thereby the expression of p40 and other relevant ICSBP target genes.
In considering the novel 12/15-LOX-dependent pathway described above as a potential therapeutic target, the Puré lab discovered that 12/15-LOX also plays a critical role in the development of hematopoietic cells and the disruption of this pathway leads to the development of a myeloproliferative disease (MPD)/myelodisplastic syndrome (MDS) that can lead to the development of myeloid leukemia. As in mature inflammatory cells (macrophages), the lack of 12/15-LOX results in the dysregulation of the IRF-8/ICSBP transcription factor and expression of its target genes. Our goals are to a) determine the mechanism by which 12/15-LOX regulates IRF-8/ICSBP function/expression, b) identify genes that cooperate with the loss of 12/15-LOX to promote progression from the chronic myeloproliferative/myelodysplastic phase to the accelerated phase and fatal blast crisis/leukemic phase in mice, and 3) determine the role of (the human homolog of) 12/15-LOX in myelogenous leukemias in man.