Targeted Cancer Therapy: The Next Generation of Cancer Treatment.

Cancer is one of the leading causes of death in the United States along with heart disease. The hallmark of cancer treatment has been conventional chemotherapy. Chemotherapeutic drugs are designed to target not only rapidly dividing cells, such as cancer cells, but also certain normal cells, such as intestinal epithelium. Over the past several years, a new generation of cancer treatment has come to the forefront, i.e, targeted cancer therapies. Like conventional chemotherapy, targeted cancer therapies use pharmacological agents that inhibit growth, increase cell death and restrict the spread of cancer. As the name suggests, targeted therapies interfere with specific proteins involved in tumorigenesis. Rather than using broad base cancer treatments, focusing on specific molecular changes which are unique to a particular cancer, targeted cancer therapies may be more therapeutically beneficial for many cancer types, including lung, colorectal, breast, lymphoma and leukemia. Moreover, recent advances have made it possible to analyze and tailor treatments to an individual patient's tumor. There are three main types of targeted cancer therapies; 1) monoclonal antibodies, 2) small molecule inhibitors and 3) immunotoxins. This review will discuss these three classes of targeted therapies in detail, as well as the biology behind targeted cancer therapies.

Cardiac fibroblast physiology and pathology.

Cardiac function is mediated by interactions between the cellular constituents of the heart, as well as the extracellular matrix. The major cell types of the heart include cardiac fibroblasts, myocytes, endothelial cells, and vascular smooth muscle cells. In addition, there are also resident stem cells and transient cell types, such as immune cells. Interactions in the heart include chemical, mechanical, and electrical signals, which vary depending on the developmental stage, disease state, and specific cell type. Understanding how these different signals interact at the molecular, cellular, and organ levels is important for better understanding cardiac function under a variety of physiological and pathological conditions. Cardiac fibroblasts play key roles in maintaining normal cardiac form and function, as well as in the cardiac remodeling process during pathological conditions, such as myocardial infarction and hypertension. Regardless of normal or pathological status of the heart, fibroblasts have multiple functions, such as synthesis and deposition of extracellular matrix and cell-cell communication with other cardiac cells, including myocytes and endothelial cells. Interactions with other cell types can affect multiple cell signaling pathways (e.g., ERK, JNK, and p38), the expression and secretion of numerous growth factors and cytokines, microRNA exchange, gene and protein expression, and angiogenesis. In this review, we provide insight into the cardiac fibroblast under normal and pathological conditions to illustrate their importance in maintaining proper cardiac function.

Dynamic cell-cell and cell-ECM interactions in the heart.

Recent studies have placed an increasing amount of emphasis on the cardiovascular system and understanding how the heart and its vasculature can be regenerated following pathological stresses, such as hypertension and myocardial infarction. The remodeling process involves the permanent cellular constituents of the heart including myocytes, fibroblasts, endothelial cells, pericytes, smooth muscle cells and stem cells. It also includes transient cell populations, such as immune cells (e.g. lymphocytes, mast cells and macrophages) and circulating stem cells. Following injury, there are dramatic shifts in the various cardiac cell populations that can affect cell-cell and cell-extracellular matrix interactions and cardiac function. Cardiac fibroblasts are a key component in normal heart function, as well as during the remodeling process through dynamic cell-cell interactions and synthesis and degradation of the extracellular matrix. Fibroblasts dynamically interact with the various cardiac cell populations through mechanical, chemical (autocrine and/or paracrine) and electrophysiological means to alter gene and protein expression, cellular processes and ultimately cardiac function. Better understanding these cell-cell and cell-extracellular matrix interactions and their biological consequences should provide novel therapeutic targets for the treatment of heart disease. In this review we discuss the nature of these interactions and the importance of these interactions in maintaining normal heart function, as well as their role in the cardiac remodeling process. This article is part of a Special Issue entitled "Myocyte-Fibroblast Signalling in Myocardium."

Analyzing cell-cell interactions in 3-dimensional adhesion assays.

The organization of cells is key to the proper formation and function of tissues and it appears to be dependent upon various intracellular and extracellular signals. These signals come from cell-cell interactions, as well as interactions with the surrounding extracellular milieu. In order to investigate these properties and interactions among cells, our lab utilizes and has developed several techniques that provide a 3-dimensional, in vivo-like environment for in vitro cell culture. In this chapter, we describe several techniques for isolating primary cardiac cells, including myocytes, endothelial cells, and fibroblasts. In addition, we discuss and outline an adhesion assay and an aggregation assay that can be used for numerous cell types, as well as a collagen gel assay for examination of cell-cell and cell-matrix interactions.

Pressure overload induces early morphological changes in the heart.

Cardiac hypertrophy, whether pathological or physiological, induces a variety of additional morphological and physiological changes in the heart, including altered contractility and hemodynamics. Events exacerbating these changes are documented during later stages of hypertrophy (usually termed pathological hypertrophy). Few studies document the morphological and physiological changes during early physiological hypertrophy. We define acute cardiac remodeling events in response to transverse aortic constriction (TAC), including temporal changes in hypertrophy, collagen deposition, capillary density, and the cell populations responsible for these changes. Cardiac hypertrophy induced by TAC in mice was detected 2 days after surgery (as measured by heart weight, myocyte width, and wall thickness) and peaked by day 7. Picrosirius staining revealed increased collagen deposition 7 days after TAC; immunostaining and flow cytometry indicated a concurrent increase in fibroblasts. The findings correlated with angiogenesis in TAC hearts; a decrease in capillary density was observed at day 2, with recovery to sham-surgery levels by day 7. Increased pericyte levels, which were observed 2 days after TAC, may mediate this angiogenic transition. Gene expression suggests a coordinated response in growth, extracellular matrix, and angiogenic factors to mediate the observed morphological changes. Our data demonstrate that morphological changes in response to cardiovascular injury occur rapidly, and the present findings allow correlation of specific events that facilitate these changes.

Cardiac myocyte-fibroblast interactions and the coronary vasculature.

Treatment of cardiovascular diseases relies on the ability not only to abrogate and compensate for congenital deformities but also to repair cardiac pathologies in the adult. Determining how cells communicate within the myocardium and how to use this communication to repair and treat pathological conditions have been necessary steps in the successful intervention of cardiac diseases. In this regard, research has mostly focused on relationships between the main cellular constituents of the heart, myocytes, and fibroblasts. However, the coronary vasculature is also critical to myocardial organization and integrity, and how the vasculature influences and responds to cues from cardiac myocytes and fibroblasts is largely underappreciated. This review discusses how factors that affect myocyte and fibroblast physiology and communication may also interact with the coronary vasculature. Defining the mechanisms of these cellular relationships will help identify ways to control angiogenesis during cardiac remodeling and the development of tissue-engineered therapies.

Deletion of periostin reduces muscular dystrophy and fibrosis in mice by modulating the transforming growth factor-ß pathway.

The muscular dystrophies are broadly classified as muscle wasting diseases with myofiber dropout due to cellular necrosis, inflammation, alterations in extracellular matrix composition, and fatty cell replacement. These events transpire and progress despite ongoing myofiber regeneration from endogenous satellite cells. The degeneration/regeneration response to muscle injury/disease is modulated by the proinflammatory cytokine transforming growth factor-ß (TGF-ß), which can also profoundly influence extracellular matrix composition through increased secretion of profibrotic proteins, such as the matricellular protein periostin. Here we show that up-regulation and secretion of periostin is pathological and enhances disease in the δ-sarcoglycan null (Sgcd(-/-)) mouse model of muscular dystrophy (MD). Indeed, MD mice lacking the Postn gene showed dramatic improvement in skeletal muscle structure and function. Mechanistically, Postn gene deletion altered TGF-ß signaling so that it now enhanced tissue regeneration with reduced levels of fibrosis. Systemic antagonism of TGF-ß with a neutralizing monoclonal antibody mitigated the beneficial effects of Postn deletion in vivo. These data suggest that periostin functions as a disease determinant in MD by promoting/allowing the pathological effects of TGF-ß, suggesting that inhibition of periostin could represent a unique treatment approach.

c-Myc is required for proper coronary vascular formation via cell- and gene-specific signaling.

OBJECTIVE: Although significant research has detailed angiogenesis during development and cancer, little is known about cardiac angiogenesis, yet it is critical for survival following pathological insult. The transcription factor c-Myc is a target of anticancer therapies because of its mitogenic and proangiogenic induction. In the current study, we investigate its role in cardiac angiogenesis in a cell-dependent and gene-specific context. METHODS AND RESULTS: Angiogenesis assays using c-Myc-deficient cardiac endothelial cells and fibroblasts demonstrate that c-Myc is essential to vessel formation, and fibroblast-mediated vessel formation is dependent on c-Myc expression in fibroblasts. Gene analyses revealed that c-Myc-mediated gene expression is unique in cardiac angiogenesis and varies in a cell-dependent manner. In vitro 3-dimensional cultures demonstrated c-Myc's role in the expression of secreted angiogenic factors, while also providing evidence for c-Myc-mediated cell-cell interactions. Additional in vivo vascular analyses support c-Myc's critical role in capillary formation and vessel patterning during development and also in response to a pathological stimulus where its expression in myocytes is required for angiogenic remodeling. CONCLUSIONS: These data demonstrate that proper c-Myc expression in cardiac fibroblasts and myocytes is essential to cardiac angiogenesis. These results have the potential for novel therapeutic applications involving the angiogenic response during cardiac remodeling.

Desmoplakin is important for proper cardiac cell-cell interactions.

Normal cardiac function is maintained through dynamic interactions of cardiac cells with each other and with the extracellular matrix. These interactions are important for remodeling during cardiac growth and pathophysiological conditions. However, the precise mechanisms of these interactions remain unclear. In this study we examined the importance of desmoplakin (DSP) in cardiac cell-cell interactions. Cell-cell communication in the heart requires the formation and preservation of cell contacts by cell adhesion junctions called desmosome-like structures. A major protein component of this complex is DSP, which plays a role in linking the cytoskeletal network to the plasma membrane. Our laboratory previously generated a polyclonal antibody (1611) against the detergent soluble fraction of cardiac fibroblast plasma membrane. In attempting to define which proteins 1611 recognizes, we performed two-dimensional electrophoresis and identified DSP as one of the major proteins recognized by 1611. Immunoprecipitation studies demonstrated that 1611 was able to directly pulldown DSP. We also demonstrate that 1611 and anti-DSP antibodies co-localize in whole heart sections. Finally, using a three-dimensional in vitro cell-cell interaction assay, we demonstrate that 1611 can inhibit cell-cell interactions. These data indicate that DSP is an important protein for cell-cell interactions and affects a variety of cellular functions, including cytokine secretion.

Cyclic 3',5'-guanosine monophosphate-dependent protein kinase inhibits colon cancer cell adaptation to hypoxia.

BACKGROUND: Type 1 cyclic 3',5'-guanosine monophosphate-dependent protein kinase (PKG) has recently been reported to inhibit tumor growth and angiogenesis. These effects suggest that PKG activation may have therapeutic value for colon cancer treatment, but the signaling downstream of this enzyme is poorly understood. The present study examined the mechanism underlying the inhibition of angiogenesis by PKG. METHODS: The effect of ectopically expressed PKG on colon cancer cell adaptation to a 1% O(2) (hypoxic) environment was examined in vitro by measuring hypoxic markers, cell death/viability, and hypoxia inducible factor (HIF) activity. RESULTS: Ectopic PKG inhibited angiogenesis in SW620 xenografts and significantly attenuated hypoxia-induced increases in vascular endothelial growth factor at both the mRNA and protein levels. PKG activation also blocked hypoxia-induced hexokinase 2 expression, which corresponded with reduced cellular adenosine triphosphate levels. Moreover, PKG expression significantly reduced cell viability and promoted necrotic cell death after 2 days in a hypoxic environment. To gain some mechanistic insight, the effect of PKG on HIF activation was determined using luciferase reporter assays. PKG activation inhibited HIF transcriptional activity in several colon cancer cell lines, including SW620, HCT116, and HT29. The mechanism by which PKG can inhibit HIF activity is not known, but it does not affect HIF-1α protein accumulation or nuclear translocation. CONCLUSIONS: These findings demonstrate for the first time that PKG can block the adaptation of colon cancer cells to hypoxia and highlights this enzyme for further evaluation as a potential target for colon cancer treatment.

Hematopoietic derived cell infiltration of the intestinal tumor microenvironment in Apc Min/+ mice.

Tumors consist of a heterogeneous population of neoplastic cells infiltrated by an equally heterogeneous collection of nonneoplastic cells that comprise the tumor microenvironment. Tumor growth, invasion, and metastasis depend on multiple interactions between these cells. To assess their potential as therapeutic targets or vehicles for tumor specific delivery of therapeutic agents, we examined the contribution of bone marrow derived cells (BMDCs) to the intestinal tumor microenvironment. Hematopoietic stem cells expressing the enhanced green fluorescent protein (eGFP) were transplanted into lethally irradiated ApcMin/+ mice, and their engraftment was analyzed by confocal microscopy. The results showed abundant infiltration of eGFP cells into the small intestine, colon, and spleen compared to heart, muscle, liver, lung, and kidney. Within the intestine, there was a pronounced gradient of engraftment along the anterior to posterior axis, with enhanced infiltration into adenomas. Immunofluorescence analysis showed that osteopontin was expressed in tumor stromal cells but not in nontumor stromal populations, suggesting that gene expression in these cells is distinct. Tumor vasculature in ApcMin/+ mice was chaotic compared to normal intestinal regions. Our data suggest that BMDCs can be harnessed for tumor-targeted therapies to enhance antitumor efficacy.

Dynamic interactions between the cellular components of the heart and the extracellular matrix.

The heart is composed of both cellular and acellular components that act in a dynamic fashion from birth to death. The cellular components consist of myocytes, fibroblasts, and vascular cells, including endothelium and smooth muscle. Changes in these components are intimately associated with function by altering the mechanical, chemical, and electrical properties of the heart. In future investigations, it will be important to examine these interactions as dynamic changes in response to physiological signals.

The dynamics of fibroblast-myocyte-capillary interactions in the heart.

In the heart, electrical, mechanical, and chemical signals create an environment essential for normal cellular responses to developmental and pathologic cues. Communication between fibroblasts, myocytes, and endothelial cells, as well as the extracellular matrix, are critical to fluctuations in heart composition and function during normal development and pathology. Recent evidence suggests that cytokines play a role in cell-cell signaling in the heart. Indeed, we find that interactions between myocytes and cardiac fibroblasts results in increased interleukin-6 and tumor necrosis factor-alpha secretion. We also used confocal and transmission electron microscopy to observe close relationships and possible direct communication between these cells in vivo. Our results highlight the importance of direct cell-cell communication in the heart, and indicate that interactions between fibroblasts, myocytes, and capillary endothelium results in differential cytokine expression. Studying these cell-cell interactions has many implications for the process of cardiac remodeling and overall heart function during development and cardiopathology.

Laying the groundwork for growth: Cell-cell and cell-ECM interactions in cardiovascular development.

Cardiac development is reliant upon the spatial and temporal regulation of both genetic and chemical signals. Central to the communication of these signals are direct interactions between cells and their surrounding environment. The extracellular matrix (ECM) plays an integral role in cell communication and tissue growth throughout development by providing both structural support and chemical signaling factors. The present review discusses elements of cell-cell and cell-ECM interactions involved in cardiogenesis, and how disruption of these interactions can result in numerous heart defects. Examining the relationships between cells and their immediate environment has implications for novel and existing therapeutic strategies to combating congenital disorders.

The extracellular matrix: at the center of it all.

The extracellular matrix is not only a scaffold that provides support for cells, but it is also involved in cell-cell interactions, proliferation and migration. The intricate relationships among the cellular and acellular components of the heart drive proper heart development, homeostasis and recovery following pathological injury. Cardiac myocytes, fibroblasts and endothelial cells differentially express and respond to particular extracellular matrix factors that contribute to cell communication and overall cardiac function. In addition, turnover and synthesis of ECM components play an important role in cardiac function. Therefore, a better understanding of these factors and their regulation would lend insight into cardiac development and pathology, and would open doors to novel targeted pharmacologic therapies. This review highlights the importance of contributions of particular cardiac cell populations and extracellular matrix factors that are critical to the development and regulation of heart function.

Fractal and image analysis of the microvasculature in normal intestinal submucosa and intestinal polyps in Apc(Min/+) mice.

Tumors are supported by the development of a unique vascular bed. We used fractal dimension (Db) and image analysis to quantify differences in the complexity of the vasculature in normal intestinal submucosa and intestinal polyps. Apc(Min/+) mice and wild-type mice were perfused with a curable latex compound, intestines sectioned, and images collected via confocal microscopy. The images were analyzed and area (A), perimeter (P), and integrated optical density (IOD) of the normal and tumor vascular beds were measured. The Db, a quantitative descriptor of morphological complexity, was significantly greater for the polyp vasculature from Apc(Min/+) mice than controls. This indicates that the polyp microvasculature is more chaotic than that of the controls, while the IOD and average vascular density values displayed no differences. This suggests the mass of blood volume is equivalent in normal and polyp microvasculature. The lower vascular area-perimeter ratios expressed by the polyp microvasculature suggest it is composed of smaller, more tortuous vessels. These data demonstrate that fractal analysis is applicable for providing a quantitative description of vascular complexity associated with angiogenesis occurring in normal or diseased tissue. Application of Db, IOD, and average density provides a clearer quantification of the complex morphology associated with tissue microvasculature.

Cardiac fibroblast: the renaissance cell.

The permanent cellular constituents of the heart include cardiac fibroblasts, myocytes, endothelial cells, and vascular smooth muscle cells. Previous studies have demonstrated that there are undulating changes in cardiac cell populations during embryonic development, through neonatal development and into the adult. Transient cell populations include lymphocytes, mast cells, and macrophages, which can interact with these permanent cell types to affect cardiac function. It has also been observed that there are marked differences in the makeup of the cardiac cell populations depending on the species, which may be important when examining myocardial remodeling. Current dogma states that the fibroblast makes up the largest cell population of the heart; however, this appears to vary for different species, especially mice. Cardiac fibroblasts play a critical role in maintaining normal cardiac function, as well as in cardiac remodeling during pathological conditions such as myocardial infarct and hypertension. These cells have numerous functions, including synthesis and deposition of extracellular matrix, cell-cell communication with myocytes, cell-cell signaling with other fibroblasts, as well as with endothelial cells. These contacts affect the electrophysiological properties, secretion of growth factors and cytokines, as well as potentiating blood vessel formation. Although a plethora of information is known about several of these processes, relatively little is understood about fibroblasts and their role in angiogenesis during development or cardiac remodeling. In this review, we provide insight into the various properties of cardiac fibroblasts that helps illustrate their importance in maintaining proper cardiac function, as well as their critical role in the remodeling heart.

The role of interleukin-6 in the formation of the coronary vasculature.

The formation and the patterning of the coronary vasculature are critical to the development and pathology of the heart. Alterations in cytokine signaling and biomechanical load can alter the vascular distribution of the vessels within the heart. Changes in the physical patterning of the vasculature can have significant impacts on the relationships of the pressure-flow network and distribution of critical growth and survival factors to the tissue. Interleukin-6 (IL-6) is a pleiotropic cytokine that regulates several biological processes, including vasculogenesis. Using both immunohistological and cardioangiographic analyses, we tested the hypothesis that IL-6-loss will result in decreased vessel density, along with changes in vascular distribution. Moreover, given the impact of vascular patterning on pressure-flow and distribution mechanics, we utilized non-Euclidean geometrical fractal analysis to quantify the changes in patterning resulting from IL-6-loss. Our analyses revealed that IL-6-loss results in a decreased capillary density and increase in intercapillary distances, but does not alter vessel size or diameter. We also observed that the IL-6-/- coronary vasculature had a marked increase in fractal dimension (D value), indicating that IL-6-loss alters vascular patterning. Characterization of IL-6-loss on coronary vasculature may lend insight into the role of IL-6 in the formation and patterning of the vascular bed.

IL-6 loss causes ventricular dysfunction, fibrosis, reduced capillary density, and dramatically alters the cell populations of the developing and adult heart.

Interleukin-6 (IL-6) is a pleiotropic cytokine responsible for many different processes including the regulation of cell growth, apoptosis, differentiation, and survival in various cell types and organs, including the heart. Recent studies have indicated that IL-6 is a critical component in the cell-cell communication between myocytes and cardiac fibroblasts. In this study, we examined the effects of IL-6 deficiency on the cardiac cell populations, cardiac function, and interactions between the cells of the heart, specifically cardiac fibroblasts and myocytes. To examine the effects of IL-6 loss on cardiac function, we used the IL-6(-/-) mouse. IL-6 deficiency caused severe cardiac dilatation, increased accumulation of interstitial collagen, and altered expression of the adhesion protein periostin. In addition, flow cytometric analyses demonstrated dramatic alterations in the cardiac cell populations of IL-6(-/-) mice compared with wild-type littermates. We observed a marked increase in the cardiac fibroblast population in IL-6(-/-) mice, whereas a concomitant decrease was observed in the other cardiac cell populations examined. Moreover, we observed increased cell proliferation and apoptosis in the developing IL-6(-/-) heart. Additionally, we observed a significant decrease in the capillary density of IL-6(-/-) hearts. To elucidate the role of IL-6 in the interactions between cardiac fibroblasts and myocytes, we performed in vitro studies and demonstrated that IL-6 deficiency attenuated the activation of the STAT3 pathway and VEGF production. Taken together, these data demonstrate that a loss of IL-6 causes cardiac dysfunction by shifting the cardiac cell populations, altering the extracellular matrix, and disrupting critical cell-cell interactions.